Three hybrid assay system

ABSTRACT

The invention provides compositions and methods for isolating ligand binding polypeptides for a user-specified ligand, and for isolating small molecule ligands for a user-specified target polypeptide using an improved class of hybrid ligand compounds.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a “continuation in part (CIP)” application ofU.S. Ser. No. 10/091,177, filed on Mar. 4, 2002, which claims priorityto U.S. Provisional applications No. 60/272,932, filed on Mar. 2, 2001;No. 60/278,233, filed on Mar. 23, 2001; and No. 60/329,437, filed onOct. 15, 2001, the specifications of which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Protein interactions facilitate most biological processesincluding signal transduction and homeostasis. The elucidation ofparticular interacting protein partners facilitating these biologicalprocesses has been advanced by the development of in vivo “two-hybrid”or “interaction trap” methods for detecting and selecting interactingprotein partners (see Fields & Song (1989) Nature 340: 245-6; Gyuris etal. (1993) Cell 75: 791-803; U.S. Pat. No. 5,468,614; and Yang et al.(1995) Nucleic Acid Research 23, 1152-1156). These methods rely upon thereconstitution of a nuclear transcriptional activator via theinteraction of two binding partner polypeptides—i.e. a first polypeptidefused to a DNA binding domain (BD) and a second polypeptide fused to atranscriptional activation domain (AD). When the first and the secondpolypeptides interact, the interaction can be detected by the activationof a reporter gene containing binding sites for the DNA binding domain.For this method to work, both proteins need to be soluble and must beable to localized to the nucleus. Accordingly, the interaction ofpolypeptides which are normally localized to other compartments may notbe detected because of the absence of other non-nuclear polypeptidecomponents which facilitate the interaction or particular non-nuclearpost-translational modifications which fail to occur in the nucleus orbecause the interacting proteins fail to fold properly when localized tothe nuclear compartment. In particular, the nuclear two-hybrid assay isill-suited to the detection of protein interactions occurring within orat the surface of cellular membranes. In addition, this assay isunsuited for screening small molecule-protein interactions because itrelies solely on genetically encoded fusion proteins.

[0003] A fundamental area of inquiry in pharmacology and medicine is thedetermination of ligand-receptor interactions. The pharmacological basisof drug action, at the cellular level, is quite often the consequence ofnon-covalent interactions between therapeutically relevant small organicmolecules and high affinity binding proteins within a specific celltype. These small organic ligands may function as agonists orantagonists of key regulatory events which orchestrate both normal andabnormal cellular functions. For years the pharmaceutical industry'sapproach to discovering such ligands has been one of the randomscreening of thousands of small molecules in specific in vitro and invivo assays to determine a potent lead compound for their drug discoveryefforts. Using these tools, a lead compound may be found to exert verywell-defined effects with regard to a function in one particular celltype (e.g. inhibition of cytokine production or DNA replication in aparticular cancer cell line). However, such results may give littleindication as to the mechanism of action at the molecular(ligand-protein interaction) level. Furthermore, the screening forpotent action on one cellular function may miss out oncross-reactivities of a lead compound giving rise to undesiredside-effects. Such side-effects often are the consequence of proteinswith closely similar structures having different functions, or of aprotein fulfilling different functions when expressed in different celltypes, or even when localized to different sub-cellular compartments.Therefore, the identification of the possibly various protein targetsfor a pharmacological agent displaying a given activity is challengingbut highly desirable. There is an unmet need for a general and efficientmethod to identify the cellular targets for these pharmacological agentsso as to accelerate the search for novel drugs both at the basic andapplied levels of research.

[0004] Similarly, there is a need for a general approach to identify asmall molecule capable of binding any selected cellular targetregardless of its biological function. Fowlkes et al. (WO 94/23025) andBroach et al. (WO 95/30012) described a screening assay for identifyingmolecules capable of binding cell surface receptors so as to activate aselected signal transduction pathway. These references describe themodification of selected yeast signaling pathways so as to mimic stepsin the mammalian signaling pathway. This latter approach is specific forcertain signaling pathways and has limited utility for broadlydiscovering small molecules that interact with any cellular target.Thus, there is also an unmet need for a general screening method todetermine the interaction between small molecules and target proteins soas to identify new drugs that are capable of specific therapeuticeffects in a variety of disease states as well as to identify agonistsand antagonists that may interfere or compete with the binding of thesmall molecules for these targets.

[0005] At this time, few (if any) efficient methodologies exist forrapidly identifying a biological target such as a protein for aparticular small molecule ligand. Existing approaches include the use ofaffinity chromatography, radio-labeled ligand binding and photoaffinitylabeling in combination with protein purification methods to detect andisolate putative target proteins. This is followed by cloning of thegene encoding the target protein based on the peptide sequence of theisolated target. These approaches require substantial re-development ofmatrices and the conditions of their use for each ligand underinvestigation, and are therefore laborious and painstaking.

[0006] Crabtree et al. (WO 94/18317) described a method to activate atarget gene in cells comprising (a) the provision of cells containingand capable of expressing (i) at least one DNA construct comprising atleast one receptor domain, capable of binding to a selected ligand,fused to a heterologous additional protein capable of initiating abiological process upon exposure of the fusion construct to the ligand,wherein the biological process comprises the expression of the targetgene, wherein the ligand is capable of binding to two or more fusionproteins, and wherein the biological process is only initiated uponbinding of the ligand to two or more fusion proteins, the two fusionproteins being the same or different, and (ii) the target gene under theexpression control of a control element which is transcriptionallyresponsive to the initiation of said biological process; and (b)exposing said cells to said ligand in an amount effective to result inexpression of the reporter gene. Further described are DNA constructs,ligands and kits useful for performing such method. Related documentsU.S. Pat. No. 5,830,462, U.S. Pat. No. 5,869,337 U.S. Pat. No. 6,165,787show these and other embodiments; specifically, Holt et al. (WO96/06097) describes the synthesis of hybrid ligands for use with thesubject methods. The purpose envisaged for these methods andcompositions is restricted to the investigation of cellular processes,the regulation of the synthesis of proteins of therapeutic oragricultural importance and the regulation of cellular processes in genetherapy. Nothing therein suggests the use of these methods andcompositions to study the interaction of proteins with small molecules,particularly in its application to pharmaceutical research and drugdevelopment.

[0007] Licitra and Liu (WO 97/41255) described a “three hybrid screenassay” in which the basic yeast two-hybrid assay system is implemented.The significant difference is: instead of depending on the interactionbetween a so-called “bait” and a so-called “prey” protein, thetranscription of the reporter gene is conditioned on the proximity ofthe two proteins, each of which can bind specifically to one of the twomoieties of a small hybrid ligand. The small hybrid ligand constitutethe “third” component of the hybrid assay system. In that system, oneknown moiety of the hybrid ligand will bind to the “bait” protein, whilethe interaction between the other moiety and the “prey” protein can beexploited to screen for either a protein that can bind a known moiety,or a small moiety (pharmaceutical compound or drug) that can bind aknown protein target.

[0008] However, the three hybrid system of Liu suffers from severallimitations: 1) the use of a transcriptional activation reporter assayis ill-suited for non-nuclear proteins, for example, membrane-boundproteins and cytosolic proteins; 2) the hybrid ligand must be localizedto the nucleus, and remains stable; and, 3) the interaction between the“bait” protein and its binding moiety on the hybrid ligand must havehigh affinity, preferably at the nanomolar level. For example,FK506-FKBP interaction was used which provides micromolar affinity.Higher affinity bewteen bait protein and its binding partner is desiredfor improving system performance.

[0009] Lin et al. (J. Am. Chem. Soc. 2000, 122:4247-8) improved upon theexisting three hybrid system by replacing the FK506-FKBP pair with ahybrid ligand consisting of dihydrofolate-reductase (DHFR) linked tomethotrexate (Mtx) (DHFR-Mtx), which provides picomolar affinity,thereby significantly improving system performance.

[0010] U.S. Pat Nos. 5,585,245 and 5,503,977 describe the “splitubiquitin” methods, which can detect protein-protein interactions by useof a ubiquitin specific protease to cleave a reporter polypeptide from afusion protein. Two fusion proteins are constructed, one consisting ofthe N-terminal half of ubiquitin and a prey protein (Nub-prey orprey-Nub), and the other consisting of the C-terminal half of ubiquitin,a bait protein and the reporter (bait-Cub-reporter). Association of preyand bait reconstitutes a ubiquitin structure recognized by the ubiquitinspecific protease, whereby the reporter is cleaved from the fusionprotein. The cleavage of the reporter from the fusion protein can bedetected by several techniques, e.g. cleavage or destabilizing thereporter or allow for its translocation.

[0011] A further working principle used in several assay systemsdeveloped to investigate protein-protein interactions is thereconstitution of an enzymatic activity from the induced spatialproximity of enzymatic fragments mediated by the interaction of twopeptides fused to these fragments. Such an assay is termed an enzymecomplementation assay. U.S. Pat. No. 6,270,964, WO 98/44350 and Wehrmanet al., Proc. Natl. Acad. Sci. U.S.A. (2002), 99:3469-3474, showexemplary methods employing this principle.

[0012] WO 93/08278, WO 98/37186, WO 01/14539 and WO 02/22826 describeyet another biological system for the investigation of protein-proteininteractions. Therein, genetic information encoding the peptides orproteins to be tested for interactions is cloned into a vectorcomprising genetic information encoding a nucleic acid binding proteinas well as the nucleic acid sequence said nucleic acid binding proteinbinds to, such that the peptides or proteins are expressed as in-framefusions with the nucleic acid binding domain. When cells are induced toexpress the fusion peptides/proteins, they will associate with thevector that encodes them. After isolation of these complexes from thecells and testing for interaction, the nucleic acid encoding interactingpeptides/proteins is easily retrieved. WO 98/37186, WO 01/14539 and WO02/22826 particularly describe systems wherein the nucleic acid bindingprotein forms a covalent bond with its recognition motif.

[0013] So called “pull-down” techniques are still frequently used in theinvestigation of protein-protein interactions. As opposed to the methodsdescribed above, these methods are carried out in vitro rather than invivo. In essence, these methods rely on immobilizing the molecularspecies for which a binding or interaction partner is sought on asurface, and subsequently passing a solution containing potentialbinding partners/interactors over this surface. A binding/interactionpartner will be retained on the solid support, while other constituentsof the solution will be washed away. In a second step, thebinding/interaction partner is isolated for further analysis, forexample by passing a solution containing an excess of a substance knownto competitively displace binding/interaction partners from themolecular species under investigation. Alternatively, the bond betweenthe molecular species under investigation and the matrix may be severedand the complex isolated from the solid support for analysis. An exampleof the use of such a technique to identify intracellular targets ofpurvalanol B, an inhibitor of CDKs, is shown in Knockaert et al. (2000),Chem. Bio. 7:411-422.

SUMMARY OF THE INVENTION

[0014] One aspect of the instant invention provides a hybrid ligandrepresented by the general formula: R1-Y-R2, wherein:

[0015] R1 represents a first ligand selected from: a steroid, retinoicacid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide,FK506, FK506 derivative, rapamycin, tetracycline, methotrexate,novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone,estrogen, progesterone, cortisone, testosterone, nickel,2,4-diaminopteridine or cyclosporin, or a derivative thereof with minorstructural modifications;

[0016] Y represents a polyethylene linker having the general formula(CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n is aninteger from 2 to 25; and,

[0017] R2 represents a user-specified second ligand different from R1selected from: a peptide, nucleic acid, carbohydrate, polysaccharide,lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene,alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon,sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether,nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt,imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol,organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide.

[0018] In one embodiment, the first ligand binds to a polypeptide. In apreferred embodiment, the binding affinity corresponds to aligand/polypeptide dissociation constant K_(D) of less than 1 μM. Inanother preferred embodiment, the first ligand is capable of forming acovalent bond with the polypeptide.

[0019] In another embodiment, X is O. In another embodiment, Y is(CH₂—O—CH₂)_(n), where n=2 to 5. In another embodiment, R1 isdexamethasone. In another embodiment, R1 is methotrexate, a methotrexatederivative, FK506, an FK506 derivative or a 2,4-diaminopteridinederivative. In a preferred embodiment, R1 is dexamethasone, Y is(CH₂OCH₂)₃, and R2 is methotrexate or a 2,4-diaminopteridine derivative.In a most preferred embodiment, R1 is methotrexate, and Y is(CH₂—O—CH₂)_(n), where n=2 to 5.

[0020] In another embodiment, R2 is a ligand chosen from: a compoundwith a known biological effect, a compound with an unknown mechanism ofaction, a compound which binds to more than one polypeptide, a drugcandidate compound, or a compound that binds to an unknown protein.

[0021] In another embodiment, R2 binds to or inhibits a kinase.

[0022] The integer n can be from 2 to 20, or 2 to 15, or 2 to 10, or 2to 5.

[0023] A related aspect of the invention provides a hybrid ligandrepresented by the general formula: R1-Y-R2, wherein:

[0024] R1 represents a first ligand selected from: a steroid, retinoicacid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide,FK506, FK506 derivative, rapamycin, tetracycline, methotrexate,novobiocin, maltose, glutathione, biotin, vitamin D, dexamethasone,estrogen, progesterone, cortisone, testosterone, nickel,2,4-diaminopteridine derivative or cyclosporin, or a derivative withminor structural modifications;

[0025] Y represents a linker; and,

[0026] R2 represents a user-specified second ligand different from R1selected from: a peptide, nucleic acid, carbohydrate, polysaccharide,lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene,alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon,sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether,nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt,imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol,organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide;

[0027] wherein R2 binds to or inhibits a kinase.

[0028] In one embodiment, the kinase is a cyclin dependent kinase. Inanother embodiment, R2 is a compound selected from Table 2, whichcontains about 600 compounds known to be able to bind to or inhibit akinase, or a derivative thereof with minor structural modifications. Inanother embodiment, Y represents a polyethylene linker having thegeneral formula (CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂,and n is an integer from 2 to 25.

[0029] Another aspect of the invention provides a fusion polypeptide,comprising segments P1, Cub-Z, and RM, in an order wherein Cub-Z iscloser to the N-terminus of the fusion polypeptide than RM, wherein 1)P1 is a ligand binding polypeptide that binds to a non-peptide ligand ofa hybrid ligand, which has the general formula R1-Y-R2, where R1 and R2are ligands, and Y is a linker, 2) Cub is a carboxy-terminal subdomainof ubiquitin, 3) Z is an amino acid residue, 4) RM is a reporter moiety.

[0030] Another aspect of the invention provides a fusion polypeptide,comprising segments P1 and Nux, wherein 1) Nux is the amino-terminalsubdomain of a wild-type ubiquitin or a reduced-associating mutantubiquitin amino-terminal subdomain, and 2) P1 is a ligand bindingpolypeptide that binds to a non-peptide ligand of a hybrid ligand, whichhas the general formula R1-Y-R2, where R1 and R2 are ligands, and Y is alinker.

[0031] In a preferred embodiment, the non-peptide ligands of the fusionproteins are: a steroid, retinoic acid, beta-lactam antibiotic,cannabinoid, nucleic acid, FK506, FK506 derivative, rapamycin,tetracycline, methotrexate, 2,4-diaminopteridine, novobiocin, maltose,glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone,cortisone, testosterone, nickel, cyclosporin, or a derivative thereofwith minor structural modifications; or a carbohydrate, polysaccharide,lipid, prostaglandin, acyl halide, alcohol, aldehyde, alkane, alkene,alkyne, alkyl, alkyl halide, alkaloid, amine, aromatic hydrocarbon,sulfonate ester, carboxylate acid, aryl halide, ester, phenol, ether,nitrile, carboxylic acid anhydride, amide, quaternary ammonium salt,imine, enamine, amine oxide, cyanohydrin, organocadmium, aldol,organometallic, aromatic hydrocarbon, nucleoside, or a nucleotide.

[0032] In another embodiment, Z is a non-methionine amino acid. Inanother embodiment, RM is: a polypeptide capable of emitting light uponexcitation, a polypeptide with an enzymatic activity, a detectable tagor a transcription factor. In another embodiment, RM is: greenfluorescent protein, URA3 or PLV.

[0033] Another aspect of the invention provides a nucleic acid encodingthe fusion polypeptide of any one of the instant invention.

[0034] In another embodiment, X is O. In another embodiment, Y is(CH₂OCH₂)₃. In another embodiment, R1 is dexamethasone, Y is (CH₂OCH₂)₃,and R2 is methotrexate or 2,4-diaminopteridine.

[0035] Another aspect of the invention provides a composition,comprising: 1) a hybrid ligand of the general formula R1-Y-R2, where R1and R2 are ligands, R1 is different from R2 and at least one of R1 andR2 is not a peptide, Y is a linker; and, 2) at least one of two fusionpolypeptides comprising: a) a first fusion polypeptide comprisingsegments P2, Cub-Z, and RM, in an order wherein Cub-Z is closer to theN-terminus of the first fusion polypeptide than RM, wherein P2 is aligand binding polypeptide that may bind to ligand R1 or R2 of thehybrid ligand, Cub is a carboxy-terminal subdomain of ubiquitin and RMis a reporter moiety, and Z is an amino acid residue; b) a second fusionpolypeptide comprising segments Nux and P1, wherein Nux is theamino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, and P1 isa ligand binding polypeptide that may bind to ligand R1 or R2 of thehybrid ligand.

[0036] A related aspect of the invention provides a composition,comprising: 1) a hybrid ligand represented by the general formula:R1-Y-R2, wherein: a) R1 represents a first ligand selected from: asteroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleicacid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose,glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone,cortisone, testosterone, nickel, or cyclosporin, or a derivative thereofwith minor structural modifications; b) Y represents a polyethylenelinker having the general formula (CH₂—X—CH₂)_(n), where X represents O,S, SO, or SO₂, and n is an integer from 2 to 25; c) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; 2) at least one fusion polypeptide selectedfrom: a) a first fusion polypeptide comprising: a ligand binding domainP1 and a domain selected from the group consisting of: a DNA bindingdomain and a transcriptional activation domain, wherein the ligandbinding domain may bind the first ligand R1; and, b) a second fusionpolypeptide comprising: a candidate ligand-binding domain P2 which maybind the user-specified ligand R2 and a domain selected from the groupconsisting of: a DNA binding domain and a transcriptional activationdomain, wherein one of the first and second fusion polypeptides containsa DNA binding domain and the other fusion polypeptide contains atranscription activation domain.

[0037] Another related aspect of the invention provides a compositioncomprising: 1) A hybrid ligand represented by the general formula:R1-Y-R2, wherein: a) R1 represents a first ligand selected from: asteroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleicacid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, 2,4-diaminopteridine derivative, novobiocin, maltose,glutathione, biotin, vitamin D, dexamethasone, estrogen, progesterone,cortisone, testosterone, nickel, or cyclosporin, or a derivative thereofwith minor structural modifications; b) Y represents a polyethylenelinker having the general formula (CH₂—X—CH₂)_(n), where X represents O,S, SO, or SO₂, and n is an integer from 2 to 25; c) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; and 2) a fusion polypeptide that includes:a) at least one ligand binding domain; and, b) a functional domainheterologous to the ligand binding domain which by itself is not capableof inducing or allowing the detection of a detectable event, but whichis capable of inducing or allowing the detection of a detectable eventwhen brought into proximity of a second functional domain.

[0038] In one embodiment, the composition is a complex. In anotherembodiment, the composition is provided in an environment chosen from: acell, a container, a kit, a solution or a growth medium.

[0039] Another aspect of the invention provides method of identifying apolypeptide sequence that binds to a user-specified ligandcomprising: 1) providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified ligand, andY is a polyethylene linker having the general formula (CH₂—X—CH₂)_(n),where X represents O, S, SO, or SO₂, and n is an integer from 2 to 25;2) introducing the hybrid ligand into a population of cells, each cellcontaining a hybrid ligand screening system including: a) a reportergene operably linked to a transcriptional regulatory sequence, saidregulatory sequence including a DNA sequence which binds to a DNAbinding domain; b) a first chimeric gene encoding a first fusionpolypeptide comprising: a ligand binding domain P1 and a domain selectedfrom a DNA binding domain or a transcriptional activation domain,wherein the ligand binding domain binds the first ligand R1; and, c) asecond chimeric gene encoding a second fusion polypeptide comprising: acandidate ligand-binding domain P2 for the user-specified ligand R2 anda domain selected from a DNA binding domain or a transcriptionalactivation domain; wherein one of the two fusion polypeptides contains aDNA binding domain and the other fusion polypeptide contains atranscription activation domain; 3) allowing the hybrid ligand to bindthe ligand binding domain of the first fusion polypeptide through thefirst ligand R1 and to contact the candidate ligand binding domain ofthe second fusion polypeptide through the user-specified ligand R2 suchthat, if R2 binds to the candidate ligand binding domain, an increase inthe level of transcription of the reporter gene occurs; 4) identifying apositive ligand binding cell in which an increase in the level oftranscription of the reporter gene has occurred; and, 5) identifying thenucleic acid sequence of the second chimeric gene encoding the candidateligand binding domain that binds to the user-specified ligand R2,thereby identifying a polypeptide sequence that binds to auser-specified ligand.

[0040] In one embodiment, the nucleic acid sequence encoding thecandidate ligand binding domain polypeptide of the second fusionpolypeptide is from a library selected from: a synthetic oligonucleotidelibrary, a cDNA library, a bacterial genomic DNA fragment library, or aeukaryotic genomic DNA fragment library.

[0041] In another embodiment, the library has about 2-10 members, orabout 10-500 members, or about 500-10,000 members, or at least 10,000members.

[0042] In another embodiment, the nucleic acid sequence that encodes thecandidate ligand binding domain polypeptide sequence represents a singleuser-selected drug target.

[0043] In another embodiment, the first ligand R1 of the hybrid ligandbinds to the ligand binding domain P1 with a high affinity. In apreferred embodiment, the binding affinity corresponds to aligand/ligand binding protein dissociation constant K_(D) of less than 1μM.

[0044] In another embodiment, the first ligand is capable of forming acovalent bond with the ligand binding domain P1.

[0045] In another embodiment, X is O. In another embodiment, Y is(CH₂—O—CH₂)_(n), where n=2 to 5. In another embodiment, R1 ismethotrexate, and Y is (CH₂—O—CH₂)_(n), n=2 to 5. In another embodiment,the reporter gene is selected from: HIS3, LEU2, TRP2, TRPI, ADE2, LYS2,URA3, CYH1, CAN1, lacZ, gfp or CAT. In another embodiment, R2 binds toor inhibits a kinase.

[0046] Another aspect of the invention provides a method of identifyinga polypeptide sequence that binds to a user-specified ligandcomprising: 1) providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified liganddifferent from R1 which binds to or inhibits a kinase, at least one ofR1 and R2 is not a peptide, and Y is a linker; 2) introducing the hybridligand into a population of cells, each cell containing a hybrid ligandscreening system including: a) a reporter gene operably linked to atranscriptional regulatory sequence, said regulatory sequence includinga DNA sequence which binds to a DNA binding domain; b) a first chimericgene encoding a first fusion polypeptide comprising: a ligand bindingdomain and a domain selected from the DNA binding domain or atranscriptional activation domain, wherein the ligand binding domainbinds the first ligand R1; and, c) a second chimeric gene encoding asecond fusion polypeptide comprising: a candidate ligand-binding domainfor the user-specified ligand R2 and a domain selected from the DNAbinding domain or the transcription activation domain; wherein one ofthe two fusion polypeptides contains a DNA binding domain and the otherfusion polypeptide contains a transcription activation domain; 3)allowing the hybrid ligand to bind the ligand binding domain of thefirst fusion polypeptide through the first ligand R1 and to contact thecandidate ligand binding domain of the second fusion polypeptide throughthe user-specified ligand R2 such that, if R2 binds to the candidateligand binding domain, an increase in the level of transcription of thereporter gene occurs; 4) identifying a positive ligand binding cell inwhich an increase in the level of transcription of the reporter gene hasoccurred; and, 5) identifying the nucleic acid sequence of the secondchimeric gene encoding the candidate ligand binding domain that binds tothe user-specified ligand R2, thereby identifying a polypeptide sequencethat binds to a user-specified ligand.

[0047] In one embodiment, the kinase is a cyclin dependent kinase. Inone embodiment, R2 is a compound selected from Table 2. In oneembodiment, Y is (CH₂—X—CH₂)_(n), n=2 to 25. In one embodiment, R1represents a first ligand selected from: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative thereof with minor structuralmodifications.

[0048] In another embodiment, the method further comprises determiningthe binding affinity of the hybrid ligand to the ligand binding domainsP1 and/or P2. In a preferred embodiment, the determination of thebinding affinity is performed by surface plasmon resonance.

[0049] In another embodiment, the method further comprises determiningthe effects of the hybrid ligand that are independent of the formationof a trimeric complex comprising the hybrid ligand, P1 and P2.

[0050] In another embodiment, the method further comprises the step of:performing at least one additional separate method to confirm that thetranscription of the reporter gene is dependent on the presence of thehybrid ligand and the ligand binding domains P1 and P2. In a preferredembodiment, said additional separate method is selected from: a halogrowth assay method or a fluorescence detection growth assay. In a mostpreferred embodiment, said additional separate method is individuallyconducted on greater than about 10, 100, 1000 or 10000 differentpositive ligand binding cell-types identified in step 4).

[0051] A related aspect of the invention provides a method ofidentifying a polypeptide sequence that binds to a user-specified ligandcomprising: providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified ligand, andY is a linker; contacting the hybrid ligand with a cultured cellcomprising: a first chimeric gene encoding a first fusion polypeptidecomprising: segments P1, Cub-Z, and RM, in an order wherein Cub-Z iscloser to the N-terminus of the first fusion polypeptide than RM,wherein P1 is a ligand binding polypeptide that binds to the firstligand R1, Cub is a carboxy-terminal subdomain of ubiquitin, Z is anon-methionine amino acid residue and RM is a reporter moiety, a secondchimeric gene encoding a second fusion polypeptide comprising: segmentsNux and P2, wherein Nux is the amino-terminal subdomain of a wild-typeubiquitin or a reduced-associating mutant ubiquitin amino-terminalsubdomain, and P2 is a candidate ligand binding polypeptide for theuser-specified ligand R2; and, a ubiquitin dependent proteolytic systemcomprising an N-end rule ubiquitin specific protease (UBP); allowing thehybrid ligand to bind the ligand binding polypeptide P1 of the firstfusion polypeptide through the first ligand R1 and to contact thecandidate ligand binding polypeptide P2 of the second fusion polypeptidethrough the user-specified ligand R2 such that, when R2 binds to thecandidate ligand binding polypeptide P2, the Nux and Cub domainsassociate to form a reconstituted ubiquitin moiety and the ubiquitinspecific protease cleaves the Cub-Z peptide bond so as to release anRM-containing fragment, said fragment being susceptible to N-end ruleubiquitin-dependent proteolytic degradation; maintaining the culturedcell under conditions wherein cleavage of the Cub-Z bond is necessaryfor growth of the cell; and, identifying the sequence of the chimericgene encoding the candidate ligand binding polypeptide P2, therebyidentifying a polypeptide sequence that binds to a user-specifiedligand.

[0052] Another related aspect of the invention provides a method ofidentifying a polypeptide sequence that binds to a user-specified ligandcomprising: providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified ligand, andY is a linker; contacting the hybrid ligand with cultured cellcomprising: a first chimeric gene encoding a first fusion polypeptidecomprising: segments Nux and PI, wherein Nux is the amino-terminalsubdomain of a wild-type ubiquitin or a reduced-associating mutantubiquitin amino-terminal subdomain, and P1 is a ligand-bindingpolypeptide for the first ligand R1, a second chimeric gene encoding asecond fusion polypeptide comprising: segments P2, Cub-Z, and RM, in anorder wherein Cub-Z is closer to the N-terminus of the second fusionpolypeptide than RM, wherein P2 is a candidate ligand-bindingpolypeptide that binds to the user-specified ligand R2, Cub is acarboxy-terminal subdomain of ubiquitin, Z is a non-methionine aminoacid residue and RM is a reporter moiety; and, a ubiquitin dependentproteolytic system comprising an N-end rule ubiquitin specific protease;allowing the hybrid ligand to bind the ligand binding polypeptide P1 ofthe first fusion polypeptide through the first ligand R1 and to contactthe candidate ligand binding polypeptide P2 of the second fusionpolypeptide through the user-specified ligand R2 such that, when R2binds to the candidate ligand binding polypeptide P2, the Nux and Cubsubdomains associate to form a reconstituted ubiquitin moiety and theubiquitin specific protease cleaves the Cub-Z peptide bond so as torelease an RM-containing fragment, said fragment being susceptible toN-end rule ubiquitin-dependent proteolytic degradation; maintaining thecultured cell under conditions wherein cleavage of the Cub-Z bond isnecessary for growth of the cell; and, identifying the sequence of thesecond chimeric gene encoding the candidate ligand binding polypeptideP2, thereby identifying a polypeptide sequence that binds to auser-specified ligand.

[0053] In one embodiment, P2 is encoded by a nucleic acid from a libraryselected from the group consisting of: a synthetic oligonucleotidelibrary, a cDNA library, a bacterial genomic DNA fragment library, and aeukaryotic genomic DNA fragment library. In another embodiment, thenucleic acid sequence that encodes the candidate ligand binding proteinsequence represents a single user-selected drug-target. In anotherembodiment, the first ligand of the hybrid ligand binds to the ligandbinding polypeptide with a high affinity. In another embodiment, thefirst ligand is methotrexate and the first ligand binding polypeptide isDHFR. In another embodiment, the binding affinity corresponds to aligand/ligand binding protein dissociation constant of less than 1 μM.In another embodiment, the first ligand is capable of forming a covalentbond with the ligand binding polypeptide. In another embodiment, Y is(CH₂OCH₂)₃. Preferably, R1 is dexamethasone, Y is (CH₂OCH₂)₃, and R2 ismethotrexate or 2,4-diaminopteridine. In another embodiment, thereporter moiety (RM) is a negative selectable marker expressed in a cellexpressing the first and second fusion polypeptides, and wherein adecrease in the level of the reporter moiety causes an increase in thegrowth of said cell. In another embodiment, the reporter moiety (RM) isa positive selectable marker expressed in a cell expressing the firstand second fusion polypeptides, and wherein a increase in the activityof the reporter moiety causes an increase in the growth of said cell.

[0054] Another related aspect of the invention provides a method ofidentifying a polypeptide sequence that binds to a user-specified ligandcomprising: providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified ligand, andY is a linker; contacting the hybrid ligand with a cultured cellcomprising: a first chimeric gene encoding a first fusion polypeptidecomprising: segments P1, Cub-Z, and RM, in an order wherein Cub-Z iscloser to the N-terminus of the first fusion polypeptide than RM,wherein P1 is a ligand binding polypeptide that binds to the firstligand R1, Cub is a carboxy-terminal subdomain of ubiquitin, Z ismethionine and RM is a reporter moiety, a second chimeric gene encodinga second fusion polypeptide comprising: segments Nux and P2, wherein Nuxis the amino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, and P2 isa candidate ligand binding polypeptide for the user-specified ligand R2;and, a ubiquitin dependent proteolytic system comprising an N-end ruleubiquitin specific protease (UBP); allowing the hybrid ligand to bindthe ligand binding polypeptide P1 of the first fusion polypeptidethrough the first ligand R1 and to contact the candidate ligand bindingpolypeptide P2 of the second fusion polypeptide through theuser-specified ligand R2 such that, when R2 binds to the candidateligand binding polypeptide P2, the Nux and Cub domains associate to forma reconstituted ubiquitin moiety and the ubiquitin specific proteasecleaves the Cub-Z peptide bond so as to release an RM-containingfragment, said fragment being non-susceptible to N-end ruleubiquitin-dependent proteolytic degradation is functional upon cleavage;maintaining the cultured cell under conditions wherein cleavage of theCub-Z bond is necessary for growth of the cell; and, identifying thesequence of the chimeric gene encoding the candidate ligand bindingpolypeptide P2, thereby identifying a polypeptide sequence that binds toa user-specified ligand.

[0055] Another aspect of the invention provides a method of determiningwhether a polypeptide P2 and a ligand R2 bind to each othercomprising: 1) translationally providing a first ligand-bindingpolypeptide comprising segments P1, Cub-Z, and RM, in an order whereinCub-Z is closer to the N-terminus of the first ligand-bindingpolypeptide than RM, and a second ligand-binding polypeptide comprisingsegments Nux and P2, wherein P1 and P2 are polypeptides, Nux is theamino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, Cub isthe carboxy-terminal subdomain of a wild-type ubiquitin, Z is an aminoacid residue and RM is a reporter moiety; 2) providing a hybrid ligandrepresented by the general formula: R1-Y-R2, wherein R1 is a firstligand that binds the first ligand-binding polypeptide at P1, R2 is asecond ligand different from R1, at least one of R1 and R2 is not apeptide, and Y is a linker; 3) allowing the hybrid ligand to contact thefirst and second ligand-binding polypeptides; 4) detecting the degree ofcleavage by a ubiquitin-specific protease (UBP) of the firstligand-binding polypeptide between Cub and Z, wherein an increase ofcleavage is indicative of polypeptide P2- ligand R2 binding.

[0056] Another aspect of the invention provides a method of determiningwhether a polypeptide P1 and a ligand R1 bind to each othercomprising: 1) translationally providing a first ligand-bindingpolypeptide comprising segments P1, Cub-Z, and RM, in an order whereinCub-Z is closer to the N-terminus of the first ligand-bindingpolypeptide than RM, and a second ligand-binding polypeptide comprisingsegments Nux and P2, wherein P1 and P2 are polypeptides, Nux is theamino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, Cub isthe carboxy-terminal subdomain of a wild-type ubiquitin, Z is an aminoacid residue and RM is a reporter moiety; 2) providing a hybrid ligandrepresented by the general formula: R1-Y-R2, wherein R1 is a firstligand, R2 is a second ligand different from R1 that binds the secondligand-binding polypeptide at P2, at least one of R1 and R2 is not apeptide, and Y is a linker; 3) allowing the hybrid ligand to contact thefirst and second ligand-binding polypeptides; 4) detecting the degree ofcleavage by a ubiquitin-specific protease (UBP) of the firstligand-binding polypeptide between Cub and Z, wherein an increase ofcleavage is indicative of protein P1- ligand R1 binding.

[0057] In one embodiment, step 1) involves the use of a cell providingan N-end rule degradation system. In one embodiment, the degree ofcleavage between Cub and Z is determined by detecting the degree ofactivity of the RM. In one embodiment, the degree of cleavage betweenCub and Z is determined by detecting the degree of enzymatic activity ofthe RM. In one embodiment, the degree of cleavage between Cub and Z isdetermined by detecting the amount of the cleaved form of RM.

[0058] Another aspect of the invention provides a method of inducing orallowing the detection of a biologically detectable event,comprising: 1) providing at least one cell comprising at least onenucleic acid sequence encoding a fusion polypeptide that includes: a) atleast one ligand binding domain; and, b) a functional domain which byitself is not capable of inducing or allowing the detection of thedetectable event; 2) providing a hybrid ligand of the general formulaR1-Y-R2, wherein R1 is different from R2, at least one of R1 and R2 isnot a peptide, R1 or R2 represents a ligand that binds to said ligandbinding domain; Y represents a polyethylene linker having the generalformula (CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n isan integer from 2 to 25; and wherein the binding of said hybrid ligandto said ligand binding domain brings the first functional domain intoproximity of a second functional domain, thereby inducing or allowingthe detection of the detectable event; and, 3) exposing said at leastone cell to an effective amount of said hybrid ligand to bring the firstfunctional domain into proximity of a second functional domain, therebyinducing or allowing the detection of the detectable event.

[0059] Another aspect of the invention provides a method of identifyinga ligand of a user-specified polypeptide, comprising: 1) providing atleast one candidate hybrid ligand having the general formula R1-Y-R2,where R1 is a first ligand, R2 is a candidate ligand, and Y is apolyethylene linker having the general formula (CH₂—X—CH₂)_(n), where Xrepresents O, S, SO, or SO₂, and n is an integer from 2 to 25; 2)introducing the candidate hybrid ligand into at least one cell whichcontains a hybrid ligand screening system including: a) a reporter geneoperably linked to a transcriptional regulatory sequence, saidregulatory sequence including a DNA sequence which binds to a DNAbinding domain; b) a first chimeric gene encoding a first fusionpolypeptide comprising: a ligand binding domain and a domain selectedfrom the DNA binding domain or a transcriptional activation domain,wherein the ligand binding domain binds the first ligand R1; and, c) asecond chimeric gene encoding a second fusion polypeptide comprising: auser-specified ligand-binding domain for the candidate ligand R2 and adomain selected from the DNA binding domain or the transcriptionactivation domain; wherein one of the two fusion polypeptides contains aDNA binding domain and the other fusion polypeptide contains atranscription activation domain; 3) allowing the candidate hybrid ligandto bind the ligand binding domain of the first fusion polypeptidethrough the first ligand R1 and to contact the user-specified ligandbinding domain of the second fusion polypeptide through the candidateligand R2 such that, if the user-specified ligand binding domain bindsto the candidate ligand R2, an increase in the level of transcription ofthe reporter gene occurs; 4) identifying the candidate hybrid ligandwhich causes an increase in the level of transcription of the reportergene in the cell, thereby identifying the candidate ligand on thecandidate hybrid ligand as a ligand for the user-specified polypeptide.

[0060] A related aspect of the invention provides a method ofidentifying a ligand that binds to a user-specified polypeptide,comprising: providing a population of candidate hybrid ligand having thegeneral formula R1-Y-R2, where R1 is a first ligand, R2 is a candidateligand, and Y is a linker; contacting each individual candidate hybridligand with a split ubiquitin hybrid ligand binding system comprising: afirst chimeric gene encoding a first fusion polypeptide comprising:segments PI, Cub-Z, and RM, in an order wherein Cub-Z is closer to theN-terminus of the first fusion polypeptide than RM, wherein P1 is aligand binding polypeptide that binds to the first ligand R1, Cub is acarboxy-terminal subdomain of ubiquitin, Z is a non-methionine aminoacid residue and RM is a reporter moiety, a second chimeric geneencoding a second fusion polypeptide comprising: segments Nux and P2,wherein Nux is the amino-terminal subdomain of a wild-type ubiquitin ora reduced-associating mutant ubiquitin amino-terminal subdomain, and P2is a user-specified polypeptide for the candidate ligand; and, aubiquitin dependent proteolytic system comprising an N-end ruleubiquitin specific protease (UBP); allowing the candidate hybrid ligandto bind the ligand binding polypeptide P1 of the first fusionpolypeptide through the first ligand R1 and to contact theuser-specified polypeptide P2 of the second fusion polypeptide throughthe candidate ligand R2 such that, when the user-specified polypeptideP2 binds to the candidate ligand R2, the Nux and Cub domains associateto form a reconstituted ubiquitin moiety and the ubiquitin specificprotease cleaves the Cub-Z peptide bond so as to release anRM-containing fragment, said fragment being susceptible to N-end ruleubiquitin-dependent proteolytic degradation; measuring the level of theRM in the presence of the candidate hybrid ligand as compared to thelevel of the RM in the absence of the hybrid ligand, wherein a decreasein the level of the RM in the presence of the hybrid ligand as comparedto the level of the RM in the absence of the hybrid ligand indicatesthat the user-specified polypeptide P2 binds to the candidate ligand R2,identifying the candidate hybrid ligand which causes a decrease in thelevel of the RM in the presence of the hybrid ligand as compared to thelevel of the RM in the absence of the hybrid ligand, thereby identifyinga ligand that binds to a user-specified polypeptide.

[0061] A related aspect of the invention provides a method ofidentifying a ligand that binds to a user-specified polypeptide,comprising: providing a population of candidate hybrid ligand having thegeneral formula R1-Y-R2, where R1 is a first ligand, R2 is a candidateligand, and Y is a linker; contacting each individual candidate hybridligand with a split ubiquitin hybrid ligand binding system comprising: afirst chimeric gene encoding a first fusion polypeptide comprising:segments Nux and P1, wherein Nux is the amino-terminal subdomain of awild-type ubiquitin or a reduced-associating mutant ubiquitinamino-terminal subdomain, and P1 is a polypeptide that binds to thefirst ligand R1 of the hybrid ligand, a second chimeric gene encoding asecond fusion polypeptide comprising: segments P2, Cub-Z, and RM, in anorder wherein Cub-Z is closer to the N-terminus of the first fusionpolypeptide than RM, wherein P2 is a user-specified ligand bindingpolypeptide for the candidate ligand R2 of the hybrid ligand, Cub is acarboxy-terminal subdomain of ubiquitin, Z is a non-methionine aminoacid residue and RM is a reporter moiety; and, a ubiquitin dependentproteolytic system comprising an N-end rule ubiquitin specific protease(UBP); allowing the candidate hybrid ligand to bind the first ligandbinding polypeptide P1 of the first fusion polypeptide through the firstligand R1 and to contact the user-specified polypeptide P2 of the secondfusion polypeptide through the candidate ligand R2 such that, when theuser-specified polypeptide P2 binds to the candidate ligand R2, the Nuxand Cub domains associate to form a reconstituted ubiquitin moiety andthe ubiquitin specific protease cleaves the Cub-Z peptide bond so as torelease an RM-containing fragment, said fragment being susceptible toN-end rule ubiquitin-dependent proteolytic degradation; measuring thelevel of the RM in the presence of the candidate hybrid ligand ascompared to the level of the RM in the absence of the hybrid ligand,wherein a decrease in the level of the RM in the presence of the hybridligand as compared to the level of the RM in the absence of the hybridligand indicates that the user-specified polypeptide P2 binds to thecandidate ligand R2, identifying the candidate hybrid ligand whichcauses a decrease in the level of the RM in the presence of the hybridligand as compared to the level of the RM in the absence of the hybridligand, thereby identifying a ligand that binds to a user-specifiedpolypeptide.

[0062] In one embodiment, P2 is encoded by a nucleic acid from a libraryselected from the group consisting of: a synthetic oligonucleotidelibrary, a cDNA library, a bacterial genomic DNA fragment library, and aeukaryotic genomic DNA fragment library. In one embodiment, the splitubiquitin hybrid ligand binding system is provided by a cell.

[0063] Another aspect of the invention provides a method to investigatethe structure activity relationship of a ligand to a ligand bindingdomain comprising: 1) providing a hybrid ligand R1-Y-R2, wherein a) R1represents a first ligand selected from: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative thereof with minor structuralmodifications; b) Y represents a polyethylene linker having the generalformula (CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n isan integer from 2 to 25; and, c) R2 represents a user-specified secondligand which is different from R1 and is selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; 2) providing cells comprising a fusionprotein that includes: a) at least one ligand binding domain; and, b) afunctional domain heterologous to the ligand binding domain which byitself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain; 3) wherein either a plurality of hybrid ligandscomprising structural variants of said second ligand R2 is provided instep 1), or a plurality of fusion proteins comprising structuralvariants of said ligand binding domain is provided in step 2); 4)exposing said cells comprising each fusion protein to an effectiveamount of each hybrid ligand such that the first functional domain maybe brought into proximity of a second functional domain thereby inducingor allowing the detection of a detectable event; 5) measuring thepresence, amount or activity of any detectable event so induced orallowed in step 4), thereby investigating the structure activityrelationship between said second ligand and the ligand binding domain.

[0064] In one embodiment, said first functional domain of (b) is chosenfrom: a DNA binding domain, a transcription activation domain, acarboxy-terminal subdomain of a wild-type ubiquitin, an amino-terminalsubdomain of a ubiquitin or a reduced-associating mutant ubiquitinamino-terminal subdomain.

[0065] Another aspect of the invention provides a method to identify ahybrid ligand having the general structure R1-Y-R2 suitable for anin-vivo assay, wherein said assay involves: 1) the use of a hybridligand, and 2) of at least one fusion polypeptide that includes: a) atleast one ligand binding domain P; and, b) a functional domain which byitself is not capable of inducing or allowing the detection of thedetectable event; and wherein said method involves the steps of: 3)synthesizing a plurality of hybrid ligands R1-Y-R2 differing by aplurality of different linkers Y, wherein R1 and R2 are different, andat least one of R1 and R2 is not a peptide; and 4) testing each hybridligand in said plurality of hybrid ligands individually for efficacy ininducing or allowing the detection of the detectable event; and 5)selecting a hybrid ligand with a particular linker that possessessuitable efficacy in inducing or allowing the detection of thedetectable event.

[0066] In one embodiment, said linker has the general structure(CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n is aninteger from 2 to 25, and the plurality of linkers differ in n. Inanother embodiment, R1 represents a first ligand selected from: asteroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleicacid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel,2,4-diaminopteridine derivative or cyclosporin, or a derivative thereofwith minor structural modifications.

[0067] Another aspect of the invention provides a kit comprising atleast one polynucleotide including a DNA fragment linked to a codingsequence for a functional domain heterologous to the DNA fragment whichby itself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain; further comprising instructions to synthesize ahybrid ligand of general structure R1-Y-R2, and to clone a ligandbinding domain into the polynucleotide, and to test the binding betweenthe hybrid ligand and the ligand binding domain, wherein R2 is differentfrom R1, one of R1 and R2 is a non-peptide ligand, and wherein one of R1and R2 binds to or inhibits a kinase.

[0068] Another aspect of the invention provides a kit comprising atleast one polynucleotide including a DNA fragment linked to a codingsequence for a functional domain heterologous to the DNA fragment whichby itself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain; further comprising instructions to synthesize ahybrid ligand of general structure R1-Y-R2, and to clone a ligandbinding domain into the polynucleotide, and to test the binding betweenthe hybrid ligand and the ligand binding domain, wherein R2 is differentfrom R1, one of R1 and R2 is a non-peptide ligand, and wherein Y is ofthe general structure (CH₂—X—CH₂)_(n), where X represents O, S, SO, orSO₂, and n is an integer from 2 to 25.

[0069] Another aspect of the invention provides a kit comprising atleast one polynucleotide including a DNA fragment linked to a codingsequence for a functional domain heterologous to the DNA fragment whichby itself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain; further comprising instructions to synthesize ahybrid ligand of general structure R1-Y-R2, and to clone a ligandbinding domain into the polynucleotide, and to test the binding betweenthe hybrid ligand and the ligand binding domain, wherein R2 is differentfrom R1, one of R1 and R2 is a non-peptide ligand, and wherein thefunctional domain is the carboxy-terminal or the amino-terminal domainof ubiquitin.

[0070] Another aspect of the invention provides a kit comprising: 1) acompound of general structure R1-Y-L, wherein Y is of the generalstructure (CH₂—X—CH₂)_(n) and L is a chemical group that is easilysubstituted by a different chemical group, and 2) instructions to usethe compound for the synthesis of a hybrid ligand R1-Y-R2 where R1 isdifferent from R2, and at least one of R1 and R2 is not a peptide.

[0071] Another aspect of the invention provides a method of doingbusiness comprising: 1) the identification of polypeptides binding to ahybrid ligand of general formula R1-Y-R2, wherein Y is of the generalstructure (CH₂—X—CH₂)_(n), R1 is different from R2, and at least one ofR1 and R2 is not a peptide, X═O, S, SO or SO₂, and wherein saidpolypeptides were previously not known to bind to such hybrid ligand,and 2) providing access to data, nucleic acids or polypeptides soobtained to another party for consideration.

[0072] In one embodiment, said identification of polypeptides isperformed using any one of the suitable methods of the instantinvention.

[0073] A related aspect of the invention provides a method of doingbusiness comprising: 1) the identification of at least one ligandbinding to a user-specified polypeptide by using a plurality of hybridligands of general formula R1-Y-R2 differing in at least one of R1 andR2, wherein R1 and R2 are ligands, R1 is different from R2, at least oneof R1 and R2 is not a peptide, Y is of the general structure(CH₂—X—CH₂)_(n), X═O, S, SO or SO₂, and wherein said ligands werepreviously not known to bind to such polypeptide, and 2) providingaccess to data and ligands obtained from such identification to anotherparty for consideration.

[0074] In a preferred embodiment, said identification of ligands isperformed using any one of the suitable methods of the instantinvention.

[0075] A related aspect of the invention provides a method to identify apolypeptide P2 which binds to a given small molecule ligand R2,comprising:

[0076] immobilizing a polypeptide P1 on a matrix, wherein thepolypeptide P1 is known to bind a ligand R1, and

[0077] contacting said matrix with a hybrid ligand of general structureR1-Y-R2, wherein Y is a linker, such that a R2-Y-R1::P1 complex isformed on the matrix, and

[0078] contacting said complex with a sample comprising at least onecandidate polypeptide P2, and

[0079] washing said surface such that all constituents of said sampleunable to bind to said complex are removed from contact with saidcomplex, and

[0080] determining whether at least one polypeptide P2 has bound to saidcomplex, and

[0081] if a polypeptide P2 was determined to bind to said complex in(v), identifying the polypeptide P2

[0082] thereby identifying a polypeptide P2 which binds to a given smallmolecule ligand R2.

[0083] In a preferred embodiment, Y is of the general structure(CH₂—X—CH₂)_(n), and n is an integer from 2 to 25 Preferably, X═O, S, SOor SO₂ It is further preferred that said sample is a mixture of severaldifferent candidate polypeptides P2, more preferably a cell extract. Inanother preferred embodiment, R1 represents a first ligand selectedfrom: steroid, retinoic acid, beta-lactam antibiotic, cannabinoid,FK506, FK506 derivative, rapamycin, tetracycline, methotrexate,novobiocin, maltose, glutathione, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, 2,4-diaminopteridine derivativeor cyclosporin, or a derivative of one of the above with minormodifications. In a further preferred embodiment, P1 is a fusionpolypeptide, comprising at least two domains which are not found incombination in nature.

[0084] In a further preferred embodiment, said fusion polypeptidecomprises

[0085] one domain chosen from the group consisting of: β-lactamase, asteroid receptor, retinoic acid receptor, cannabinoid receptor, FKB 12,Tet-R, DHFR, GyrB, maltose binding protein, glutathione-S-transferase,vitamin D receptor, glucocorticoid receptor, estrogen receptor,progesterone receptor, testosterone receptor, or a fragment of one ofthe above retaining the binding capacity to its respective ligand, and

[0086] a second domain comprising a tag which allows the immobilizationof said fusion protein on a matrix.

[0087] Preferably, said tag is chosen from the group consisting of:strep tag, FLAG tag, his₆-tag, CBD tag, E tag, GFP tag, GST tag,haemagglutinin tag, Myc tag, T7 tag, Tag 100, V5 tag, Calmodulin bindingpeptide tag, S tag, Intein/chitin binding domain tag, Xpress tag,thioredoxin tag or VSV tag. In another related embodiment of saidmethod, P2 is a fusion polypeptide comprising a user specified fragmentand a DNA-binding domain. Preferably, P2 is bound, through saidDNA-binding domain, to a DNA molecule encoding P2. Preferably, said DNAbinding domain is chosen from the group consisting of: a lac repressorprotein, a Rep protein, an NS1 or H-1 protein, a phi-29 terminalprotein, a 55 Kd protein, and fragments and derivatives of one of theabove, which fragments and derivatives retain the respective DNA bindingactivity.

[0088] A related aspect of the present invention provides a compositioncomprising:

[0089] a hybrid ligand of general structure R1-Y-R2, wherein Y is alinker, and

[0090] a fusion polypeptide, comprising at least two domains which arenot found in combination in nature, wherein

[0091] one domain is a user specified polypeptide, and

[0092] a second domain is a DNA binding domain.

[0093] Preferably, Y is of the general structure (CH₂—X—CH₂)_(n), and nis an integer from 2 to 25. More preferably, X═O, S, SO or SO₂. Inanother preferred embodimant, R1 represents a first ligand selectedfrom: steroid, retinoic acid, beta-lactam antibiotic, cannabinoid,FK506, FK506 derivative, rapamycin, tetracycline, methotrexate,novobiocin, maltose, glutathione, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, 2,4-diaminopteridine derivativeor cyclosporin, or a derivative of one of the above with minormodifications.

[0094] A related aspect of the present invention provides a method ofdetermining whether a polypeptide P2 and a ligand R2 bind to each othercomprising:

[0095] translationally providing a first ligand-binding polypeptidecomprising segments P1 and a first fragment of a β-lactamase, and asecond ligand-binding polypeptide comprising segments P2 and a secondfragment of a β-lactamase, wherein said first and second fragments of aβ-lactamase individually possess no β-lactamase activity, and whereinthe enzymatic activity of a β-lactamase is reconstituted when P1 and P2are brought into close spatial proximity; and

[0096] providing a hybrid ligand represented by the general formula:R1-Y-R2, wherein R1 is a first ligand that binds the firstligand-binding polypeptide at P1, R2 is a second ligand different fromR1, at least one of R1 and R2 is not a peptide, and Y is a linker; and

[0097] allowing the hybrid ligand to contact the first and secondligand-binding polypeptides; and

[0098] detecting the activity of a β-lactamase, wherein an increase inβ-lactamase activity in the presence of said hybrid ligand compared toin its absence is indicative of polypeptide P2—ligand R2 binding,

[0099] thereby determining whether the polypeptide P2 and a ligand R2bind to each other.

[0100] Preferably, Y is of the general structure (CH₂—X—CH₂)_(n), and nis an integer from 2 to 25. More preferably, X═O, S, SO or SO₂. Inanother preferred embodiment, R1 represents a first ligand selectedfrom: steroid, retinoic acid, beta-lactam antibiotic, cannabinoid,nucleic acid, polypeptide, FK506, FK506 derivative, rapamycin,tetracycline, methotrexate, novobiocin, maltose, glutathione, biotin,vitamin D, dexamethasone, estrogen, progesterone, cortisone,testosterone, nickel, 2,4-diaminopteridine derivative or cyclosporin, ora derivative of one of the above with minor modifications.

[0101] A related aspect of the present invention provides a compositioncomprising

[0102] a nucleic acid encoding a polypeptide comprising a fragment of aβ-lactamase and a user specified polypeptide, and

[0103] a second nucleic acid encoding a polypeptide comprising a secondfragment of a β-lactamase and a domain chosen from the group consistingof: β-lactamase, a steroid receptor, retinoic acid receptor, cannabinoidreceptor, FKB 12, Tet-R, DHFR, GyrB, maltose binding protein,glutathione-S-transferase, vitamin D receptor, glucocorticoid receptor,estrogen receptor, progesterone receptor, testosterone receptor, or afragment of one of the above, which fragment retains the bindingcapacity to its respective ligand,

[0104] wherein said first and second fragments of a β-lactamaseindividually possess no β-lactamase activity, and wherein the enzymaticactivity of a β-lactamase is reconstituted when P1 and P2 are broughtinto close spatial proximity. In a preferred ambodiment, saidcomposition further comprises a hybrid ligand of general structureR1-Y-R2, wherein Y is a linker. Preferably, Y is of the generalstructure (CH₂—X—CH₂)_(n), and n is an integer from 2 to 25. Morepreferably, X═O, S, SO or SO₂. In another preferred ambodiment, R1represents a first ligand selected from: steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative of one of the above withminor modifications.

[0105] A related aspect of the present invention provides a compositioncomprising

[0106] a polypeptide comprising a fragment of a β-lactamase and a userspecified polypeptide, and

[0107] a second polypeptide comprising a second fragment of aβ-lactamase and a domain chosen from the group consisting of:β-lactamase, a steroid receptor, retinoic acid receptor, cannabinoidreceptor, FKB12, Tet-R, DHFR, GyrB, maltose binding protein,glutathione-S-transferase, vitamin D receptor, glucocorticoid receptor,estrogen receptor, progesterone receptor, testosterone receptor, or afragment of one of the above, which fragment retains the bindingcapacity to its respective ligand,

[0108] wherein said first and second fragments of a β-lactamaseindividually possess no β-lactamase activity, and wherein the enzymaticactivity of a β-lactamase is reconstituted when P1 and P2 are broughtinto close spatial proximity. Preferably, said composition furthercomprises a hybrid, ligand of general structure R1-Y-R2, wherein Y is alinker. Preferably, Y is of the general structure (CH₂—X—CH₂)_(n), and nis an integer from 2 to 25. More preferably, X═O, S, SO or SO₂.

[0109] In another preferred embodiment, R1 represents a first ligandselected from the group consisting of: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative of one of the above withminor modifications.

BRIEF DESCRIPTION OF THE FIGURES

[0110]FIG. 1. Synthetic schemes and structure representations for GPC285937, 285985, 286004, 286026 and 285993 andMtx-(CH₂—O—CH₂)₅-purvalanol B.

[0111]FIG. 2. Sensorgram and subsequent determination of dissociationconstant K_(D) for binding of the complex Cyclin Dependent Kinase (CDK)4/Cyclin D1 (CDK4/D1) to a Methotrexate-based hybrid ligand using aBiacore 2000-SPR Biosensor. DHFR was covalently coupled to the surfaceof an SPR chip and the hybrid ligand (GPC 285985) was allowed to bind.Subsequently, solutions of different concentrations of the CDK4/D1complex (shown by different curves) were pumped over the chip surfacefor 300 sec, followed by running buffer to monitor dissociation. Thebinding characteristics of methotrexate to DHFR were taken into accountto estimate k_(ass) and k_(diss) of the hybrid ligand to CDK4/D1 and theK_(D) calculated.

[0112]FIG. 3. Structural representations of GPC 285937, GPC 285985 andGPC 285993.

[0113]FIG. 4. An example of a Halo Growth Assay. A visible halo of yeastcellular growth on medium lacking histidine indicates activation of thereporter HIS3 gene caused by the dimerization of the LexBD-DHFR andGalAD-GR2 fusion proteins in the presence of GPC 285937, but not in thepresence of DMSO alone.

[0114]FIG. 5. Activation of the HIS3 reporter gene by compound induceddimerization of the LexA-BD-DHFR and Gal4-AD-GR2 fusion proteins in thepresence of a hybrid ligand of the invention (GPC 285937) compared to aprior art hybrid ligand Mtx-mdbt-Dex (mdbt: metadibenzothioester).Microscope images of growth media where circular objects are individualyeast cells and dark woolly threads are precipitated Mtx-mdbt-Dex.Precipitation of Mtx-mdbt-Dex is seen at 100 μM.

[0115]FIG. 6. Influence of different linker moieties of hybrid ligandsand their biological effects. A hybrid ligand of the invention (GPC285937) employs 3 ethylenglycol (EG) groups as a linker, which offersimproved superiority over the metadibenzothioester linker present in theprior art hybrid ligand Mtx-mdbt-Dex by promoting better overall growthof the colony.

[0116]FIG. 7. Difference in growth of yeast colonies on screening platesin the presence of either GPC 285937 or Mtx-mdbt-Dex. Colonies growingon media with Mtx-mdbt-Dex were hardly detectable, whereas clones grewvisibly better on media containing GPC 285937.

[0117]FIG. 8. Growth curves of yeast cultures exposed to differentconcentrations of the hybrid ligand GPC 285985 in medium lackinghistidine as measured by oxygen consumption using an OxoPlate (PreSens,Germany). Yeast cultures expressing the CDK2 fusion protein show typicalgrowth curves over time. In contrast, yeast cultures expressing a CDK4fusion protein only show growth at the high concentrations of the hybridligand, confirming the specificity of the hybrid ligand to CDK2.

[0118]FIG. 9. A representation of the fusion protein Sec62-DHFR-Cub-PLVattached to the membrane of endoplasmic reticulum (ER). Whilst tetheredto the membrane, the PLV transcription factor is unable to activate areporter gene. However, on cleavage of the Cub-PLV following theformation of a quasi-native ubiquitin molecule, the cleaved PLV reportermoiety is able to shuttle to the nucleus and activate an appropriatereporter gene.

[0119]FIG. 10: A test of the hybrid ligand GPC 285985 using a yeastthree-hybrid system in a halo assay. The top row shows the growth ofcells transformed with pBTM118c-DHRF and either pGAD426c-hCDK2 (topleft) or pGAD426c-hCDK4 (top right) after two days on medium lackingtrp, leu and his following the addition of 1 μl of a 1 mM DMSO solutionGPC 285985. The bottom row shows growth after two days on medium lackingtrp and leu his following the addition of GPC 285985. On the mediumlacking histidine, only cells transformed with pGAD426c-hCDK2 displaydetectable growth, while on medium lacking only trp and leu, bothpGAD426c-hCDK2 (bottom left) and pGAD426c-hCDK4 (bottom right)transformed cells form dense populations.

[0120]FIG. 11: Weak interactions can be detected after longer periods ofgrowth. In an experiment analogous to the experiment shown in FIG. 10,cells transformed with pBTM 118c-DHRF and either pGAD426c-hCDK2 (leftpanel) or pGAD526c-hCDK4 (right panel) were incubated for six days at30° C. on medium lacking trp, leu and his after addition of 1 μl of a 1mM solution of GPC 285985 dissolved in DMSO to the center of each petridish. After this incubation time the low affinity interaction (900 μM)between CDK4 and GPC 285985 was able to allow weak but detectablegrowth. In contrast, cells expressing the CDK2 fusion protein formeddense populations under the same conditions.

[0121]FIG. 12: Results of a high throughout halo assay using clonesrecovered from a three-hybrid genetic screen. A library of fusionproteins was screened to isolated genes that encoded proteins, whichbound to the hybrid ligand GPC 285985. The table shows a sample of theanalysis performed on 2811 initial positive clones. 102 clones showedcompound specific growth. The identity of all clones was confirmed bysequencing and contained genes encoding CDK2 and other genes.

[0122]FIG. 13: An isolated plasmid coding for protein GPC761 expressedas a fusion protein with GAL4 AD (isolated from a three-hybrid geneticscreen) was co-transformed with pBTM118c-DHFR into yeast strain L40. Ahalo assay was conducted to validate and further characterize andinvestigate the structure activity relationship between the interactionbetween this protein and the hybrid ligand used for the initial screen.Only hybrid ligand comprising the active CDK2 inhibitor GPC 285985 (leftpanel) allowed growth of cells on medium lacking trp, leu and his, whilethe structural variant GPC 285993 (which does not bind to CDK2) wasineffective at promoting growth in this assay and hence did not bind toprotein GPC761.

[0123]FIG. 14: The performance of the hybrid ligands of the invention inmammalian cells was tested as described in example 11. The CAT reportergene is activate as shown by the presence of a colored precipitate inthe positive control (FIG. 14A). Cells expressing the DHFR and GR2fusions incubated with the respective dimerizing hybrid ligand GPC285937 (FIG. 14B) also show a colored precipitate, but not where GPC285937 is missing (FIG. 14C).

[0124]FIG. 15. Three-hybrid assay system based on Spit ubiquitin proteinsensor technology. Two fusion proteins are constructed, one consistingof the N-terminal half of ubiquitin (Nub) and a prey protein (XY), andthe other consisting of the C-terminal half of ubiquitin (Cub), a baitprotein (DHFR) and the reporter moiety (R). Association of prey and baitvia mutual binding to the hybrid small molecule mtx-xy reconstitutes aquasi-native ubiquitin structure (UBI) recognized by the ubiquitinspecific protease (UBPs), whereby the reporter moiety is cleaved fromthe fusion protein. The cleavage of the reporter moiety from the fusionprotein can be detected by several techniques, e.g., without limitation,Western Blot, cleavage or destabilization of the reporter via N-end ruleconsiderations (R having a non-methionine amino acid at its N-terminus)or by providing a transcription factor as R and allowing for itstranslocation into the nucleus.

[0125]FIG. 16. Effects of linker length (number of PEG repeats in thelinker) on functionality as measured by biological activity in athree-hybrid halo assay. Yeast halo growth was only seen in cells in thepresence of GPC 286026 (5 PEG units as a linker) but not in the presenceof GPC 286004 (3 PEG units as linker).

[0126]FIG. 17. Description of plasmid pACT2; a human fetal brain cDNAlibrary was obtained commercially from Clontech that was cloned in thisvector and used subsequently in screening experiments. a. A vector map.b. A restriction map and multiple cloning site.

DETAILED DESCRIPTION OF THE INVENTION

[0127] 1. Overview

[0128] In general the invention provides a three hybrid assay system andreagents for the identification of the protein binding partner of aselected small pharmaceutical agent. Likewise, the invention alsoprovides methods and reagents for the identification of a smallpharmaceutical agent binding partner of a selected protein. Oncedetected, the invention further provides methods for monitoring theinteraction of the pharmaceutical agent and its protein binding partnerthat can be used to detect competitors of the interaction.

[0129] According to one aspect of the invention, a compound binding to aknown target polypeptide can be selected from a pool/library ofcandidate compounds. Preferably, the compound is a small molecule (seedefinition below). In this aspect of the invention, each candidate smallmolecule (designated “R2” hereafter) is linked to a known small molecule(designated “R1” hereafter) via a linker sequence (designated “Y”hereafter). The resulting R1-Y-R2 compound is then allowed to contact afusion polypeptide P1-RS1, comprising the known polypeptide bindingpartner of R1, P1, fused to a first part of a reporter system (RS), RS1,and the target polypeptide (designated “P2” hereafter) fused to a secondpart of RS, RS2, in a suitable environment (such as a cell). The RS isdesigned such that when RS1 and RS2 are brought into spatial proximityin a suitable environment, the RS is activated and triggers abiologically detectable event. If R2 interacts with P2 with strongenough affinity, then RS1 is brought into close vicinity with RS2 viathe bridging effect of the R1-Y-R2 hybrid, thereby triggering theactivation of RS. Hence, contacting the environment (i.e., a cell)containing the RS, the P1—RS1-hybrid and the P2-RS2-hybrid with apool/library of R1-Y-R2-hybrids and observing activation of RSfacilitates the isolation of R1-Y-R2-hybrids, wherein R2 is able tospecifically bind to P2.

[0130] In one embodiment, the RS is a transcription-based reportersystem, such as yeast two-hybrid system. In another related embodiment,the RS is a split ubiquitin based reporter system.

[0131] In one embodiment, the linker sequence is particularly suitablefor in vivo use of the chemical compound due to its increased solubilityand enhanced membrane permeability.

[0132] In one embodiment, the P1-R1 interaction is a non-covalentinteraction. In an alternative embodiment, the P1-R1 interaction resultsin a covalent bond.

[0133] In one embodiment, the chemical library is synthesized. Inanother embodiment, the chemical library is from natural sources.

[0134] According to another aspect of the invention, a polypeptidebinding to a known target small molecule R2 can be selected from alibrary/libraries of test polypeptides. In this aspect, the target smallmolecule R2 is linked by a linker sequence Y to a known small moleculeR1 to form an R1-Y-R2 hybrid compound, which is then allowed to contactpolypeptide P1, the known binding partner of known small molecule R1,fused to RS1, in a suitable environment. A library or libraries of testpolypeptides P2, each fused to RS2, are translationally provided to thesame environment. Binding between the target small molecule R2 and anymember polypeptide P2 of the library/libraries will bring the P2-RS2hybrid into the vicinity of the P1-RS1-hybrid, thereby triggering theactivation of a reporter system RS. Hence, contacting cells containingthe RS, the P1-RS 1-hybrid and a pool/library of P2-RS2-hybrids with theR1-Y-R2-hybrid and observing activation of RS facilitates the isolationof P2-RS2-hybrids, wherein P2 is able to specifically bind to R2.

[0135] In one embodiment, the RS is a transcription-based reportersystem, such as yeast two-hybrid system. In another related embodiment,the RS is a split ubiquitin based reporter system.

[0136] In one embodiment, the linker sequence is particularly suitablefor in vivo use of the chemical compound due to its increased solubilityand enhanced membrane permeability.

[0137] In one embodiment, the P1-R1 interaction is a non-covalentinteraction. In a related embodiment, the P1-R1 interaction results in acovalent bond.

[0138] In one embodiment, the polypeptide library is cDNA library orgenomic DNA library. In another embodiment, the polypeptide library issynthesized randomly or semi-randomly. The library may contain differentnumber of members, preferably from 2 to 10 members, or 10 to 500members, 500 to 10,000 members or more than 10,000 members.

[0139] The above described methods are not only suitable to identify anunknown member of a polypeptide—ligand pair (screen method), but alsosuitable to determine if a given polypeptide binds a given ligand (assayor test method).

[0140] According to yet another aspect of the invention, there isprovided a kit for detecting and/or selecting interactions betweenpolypeptides and small molecules using either one of the above mentionedmethods.

[0141] According to another aspect of the invention, there is provided amethod for pharmaceutical research wherein interactions betweenpolypeptides and small molecules are monitored to facilitate furthercharacterization and/or optimization of binding of at least one of theidentified binding partners. This can be useful in a variety ofsituations. For example, many drugs or chemical compounds havenoticeable, sometimes even severe, undesirable side-effects. This islikely caused by the fact that the drug may non-discriminately bindproteins other than the intended target. The instant invention providesa method to identify all potential binding partners of a given drug orchemical compound, thereby providing a basis to design other relateddrugs that do not bind these non-intended targets to avoid thenondesirable side-effects. In other cases, a drug may have some efficacyfor certain conditions, but the mechanism of action of the drug isunknown, thus, it is difficult to optimize the drug for a betterefficacy. The instant invention provides a method to identify the targetof the drug, thereby offering a means to further study the biology andthe related signaling pathways so that drug optimization can be achievedbased on knowledge gained through research on those signaling pathways.Furthermore, information on the binding of ligands to polypeptide ligandbinding domains that is collected by practicing the methods of theinvention may be used to understand or further understand the functionor side effects of a ligand in a biological or therapeutic setting.Information thus collected may for example, be used to provide moreinformed prescription of medicaments comprising the ligand or withappropriate additional medicaments to provide more effective combinationtherapies. Thus, the instant invention can be used to identify orproduce any one or more of the following: a compound with a knownbiological effect, a compound with an unknown mechanism of action, acompound which binds to more than one polypeptide, a drug candidatecompound, or a compound that binds to an unknown protein.

[0142] The instant invention also provides hybrid ligands which binds toor inhibits a kinase. For example, R2 can be a compound chosen fromTable 2, which is a list of compounds that is known to bind or inhibitkinases, or a derivative thereof with minor structural modifications. Atypical kinase target can be a cyclin-dependent kinase.

[0143] Furthermore, the instant invention also provides a method toidentify novel modulators of certain known proteins and a method toproduce pharmaceutical formulations of such modulators.

[0144] Another aspect of the invention provides a method to identify acompound which inhibits the interaction between a ligand and apolypeptide, wherein the interaction is identified using any suitablemethod of the instant invention, comprising: 1) identifying, by any oneof the suitable methods of the instant invention, a polypeptide thatinteracts with a user-specified ligand, or identifying a ligand thatinteracts with a user-specified polypeptide; 2) providing an environmentwherein said interaction occurs; 3) contacting the environment with atest compound; 4) determining if said test compound inhibits saidinteraction, thereby identifying a compound which inhibits theinteraction between a ligand and a polypeptide.

[0145] In one embodiment, the ligand is a non-peptide ligand. In apreferred embodiment, the ligand is of the general structure R1-Y-R2,wherein R1, Y, and R2 are as defined above.

[0146] In one embodiment, the test compound is from a variegatedlibrary, which, for example, can be a nucleic acid library (cDNA,genomic DNA, EST, etc.) encoding polypeptides; a polypeptide library(synthetic, natural, random, semi-random, etc.); a small chemicallibrary (natural, synthetic, etc.).

[0147] In one embodiment, the environment is a cell. In a relatedembodiment, the environment contains any one of the suitable hybridligand screening system of the instant invention (including reportersystems).

[0148] The inhibitory effect of the test compound can be assessed basedon the change of status of the reporter system (see detaileddescriptions below).

[0149] This method can be useful in a variety of situations. Forexample, if a small chemical compound is initially identified aspossessing certain biological activity when administered to a cell, itsprotein target(s) can be identified. In case that multiple targets arepresent and only one target interaction is desired (for example, othertarget protein interactions lead to undesirable side effects), a testcompound can be identified using this method so that it may specificallyblocks those undesirable interactions while still allow the intendedinteraction to occur. In another scenario, after the identification ofthe polypeptide target of a known ligand, a compound can be identifiedusing the subject method to block the interaction between such ligandand polypeptide, either to eliminate the undesirable effect ofligand-polypeptide interaction, or to reversibly control suchinteraction.

[0150] Another aspect of the invention provides a method to identify apolypeptide sequence that binds to a user-specified ligand,comprising: 1) providing a hybrid ligand with the general structureR-Y-R, wherein R is a user-specified ligand and Y is a linker,preferably a linker having the general formula (—CH₂—X—CH₂—)_(n),wherein X and n are as defined above; 2) introducing the hybrid ligandinto a population of cells, each cell containing a ligand screeningsystem as defined above, or a Nux-Cub split ubiquitin-based system asdefined above, wherein both P1 and P2 (as defined above) represent thesame test polypeptide; 3) allowing the hybrid ligand to contact P1 andP2 in said ligand screening system, 4) identifying a positive ligandbinding cell in which a detectable change in the status of the reportersystem of the ligand screen system occurs; thereby identifying a nucleicacid encoding the test polypeptide.

[0151] In a related aspect of the invention, there is provided a methodto determine if a ligand binds to a polypeptide, comprising: 1)providing a hybrid ligand with the general structure R-Y-R, wherein R isa user-specified ligand and Y is a linker, preferably a linker havingthe general formula (—CH₂—X—CH₂—)_(n), wherein X and n are as definedabove; 2) introducing the hybrid ligand into an environment containing atest polypeptide, wherein multimerization (preferably dimerization) ofthe polypeptide lead to a detectable change; 3) determining if saiddetectable change occur, thereby determining if the ligand binds to thetest polypeptide.

[0152] In a related aspect, a similar method can be used to determine ifa known polypeptide interacts with a test hybrid ligand.

[0153] In one embodiment, the detectable change is an enzymatic activityof the test polypeptide, which activity is only present when saidpolypeptide is multimerized (for example, dimerized). In a relatedembodiment, the polypeptide can be linked to any one of the suitablehybrid ligand screen system described above so that multimerization ofthe polypeptide by the hybrid ligand lead to the activation of thereporter system.

[0154] In one embodiment, the polypeptide is an enzyme that is inactiveas a monomer, and is only activated as a multimer, preferably a dimer.In this embodiment, it may suffice to use only a single polynucleotidein a method of the invention. For example, where one is searching for anew ligand for a polypeptide of interest for which a ligand is alreadyknown, one could use a polynucleotide encoding the polypeptide ofinterest fused to an enzyme that is active only as a multimer,preferably a dimer, and which does not dimerize spontaneously (e.g. areduced affinity mutant). If this fusion polypeptide is contacted with ahybrid ligand R1-Y-R2 of the invention, where R1 is the known ligand forthe polypeptide of interest, and R2 is a test ligand, activity of theenzyme will only be manifest if the test ligand binds the polypeptide ofinterest.

[0155] In one embodiment, the environment is a cell.

[0156] In one embodiment, the polypeptide comprises a receptor,preferably a receptor that requires multimerization to be functional oractivated, such as a receptor that contains a cytoplasmic domain fromone of the various cell surface membrane receptors as described in WO94/18317. For example, many of these domains are tyrosine kinases or arecomplexed with tyrosine kinases, e.g. CD3 ζ, IL-2R, IL-3R, etc. For areview see Cantley, et al., Cell (1991) 64, 281. Tyrosine kinasereceptors which are activated by cross-linking, e.g. dimerization (basedon nomenclature first proposed by Yarden and Ulrich, Annit. Rev.Bioclin. (1988) 57, 443,include subclass 1: EGF-R, ATR2/neu, HER2/neu,HER3/c-erbB-3, Xmrk; subclass II: insulin-R, IGF R insulin-like growthfactor receptor], IRR; subclass III: PDGF-R-A, PDGF-R-B, CSF R(M-CSF/c-Fms), c-kit, STK-1/Flk-2; and subclass IV: FGF-R, fig [acidicFGFJ, bek [basic FGF]); neurotrophic tryosine kinases: Trk family,includes NGF-R, Ror1,2. Receptors which associate with tyrosine kinasesupon cross-linking include the CD3 ζ-family: CD3 ζ and CD3 η (foundprimarily in T cells, associates with Fyn) β and -γ chains of Fcε RI(found primarily in mast cells and basophils); γ chain of Fcγ RIII/CD16(found primarily in macrophages, neutrophils and natural killer cells);CD3 γ, δ, and ε (found primarily in T cells); Ig-a/MB-1 andIg-P/B29(found primarily in B cell). Alternatively, a cytokine-receptormay be utilized to detect ligand and receptor interactions as describedin Eyckernan et al (Nature Cell Biology 2001; 3: 1114-1119).

[0157] 2. Definitions

[0158] The term “agonist”, as used herein, is meant to refer to an agentthat mimics or up-regulates (e.g. potentiates or supplements) thebioactivity of a protein of interest, or an agent that facilitates orpromotes (e.g. potentiates or supplements) an interaction amongpolypeptides or between a polypeptide and another molecule (e.g. asteroid, hormone, nucleic acids, small molecules etc.). An agonist canbe a wild-type protein or derivative thereof having at least onebioactivity of the wild-type protein. An agonist can also be a smallmolecule that up-regulates the expression of a gene or which increasesat least one bioactivity of a protein. An agonist can also be a proteinor small molecule which increases the interaction of a polypeptide ofinterest with another molecule, e.g. a target peptide or nucleic acid.

[0159] “Antagonist” as used herein is meant to refer to an agent thatdown-regulates (e.g. suppresses or inhibits) the bioactivity of aprotein of interest, or an agent that inhibits/suppresses or reduces(e.g. destabilizes or decreases) interaction among polypeptides or othermolecules (e.g. steroids, hormones, nucleic acids, etc.). An antagonistcan be a compound which inhibits or decreases the interaction between aprotein and another molecule, e.g., a target peptide, such asinteraction between ubiquitin and its substrate. An antagonist can alsobe a compound that down-regulates the expression of a gene of interestor which reduces the amount of the wild type protein present. An agonistcan also be a protein or small molecule which decreases or inhibits theinteraction of a polypeptide of interest with another molecule, e.g. atarget peptide or nucleic acid.

[0160] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and/or insertions of nucleotides. Anallele of a gene can also be a form of a gene containing mutations.

[0161] The term “biologically detectable event” is a general term usedto describe any biological event that can be detected in an assaysystem, such as for example, without limitation, in atranscription-based yeast two hybrid assay, a split ubiquitin assay,etc. A biologically detectable event means an event that changes ameasurable property of a biological system, for example, withoutlimitation, light absorbance at a certain wavelength, light emissionafter stimulation, presence/absence of a certain molecular moiety in thesystem, electrical resistance/capacitance etc., which event isconditional on another, possibly non-measurable or less easilymeasurable property of interest of the biological system, for example,without limitation, the presence or absence of an interaction betweentwo proteins. Preferably, the change in the measurable property broughtabout by the biologically detectable event is large compared to naturalvariations in the measurable property of the system. Examples includethe yellow color resultant from the action of β-galactosidase ono-nitrophenyl-b-D-galactopyranoside (ONPG) (J. H. Miller, Experiments inMolecular Genetics, 1972) triggered by transcriptional activation of theE. coli lacZ gene encoding β-galactosidase by reconstitution of atranscription factor upon binding of two proteins fused to the twofunctional domains of the transcription factor. Other examples ofbiologically detectable events are readily apparent to the personskilled in the art. Alternatively, other biological functions may beinduced and detected following oligomerization, preferable dimerization,of the functional domains. For example, transcriptional regulation,secondary modification, cell localization, excocytosis, cell signaling,protein degradation or inactivation, cell viability, regulatedapoptosis, growth rate, cell size. Such biological events may also becontrolled by a variety of direct and indirect means includingparticular activities associated with individual proteins such asprotein kinase or phosphatase activity, reductase activity,cyclooxygenase activity, protease activity or any other enzymaticreaction dependent on subunit association. Also, one may provide forassociation of G proteins with a receptor protein associated with thecell cycle, e.g. cyclins and cdc kinases, or multiunit detoxifyingenzymes.

[0162] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means a catalytic, effector, antigenic, molecular tagging ormolecular interaction function that is directly or indirectly performedby a polypeptide (whether in its native or denatured conformation), orby any subsequence thereof.

[0163] The terms “cell death”, “cell killing” or “necrosis” refer to thephenomenon of cells dying as a result of an extrinsically imposed lossof a particular cellular function essential for the survival of thecell.

[0164] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto a particular subject cell but to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0165] “Characterize” as used herein means a detailed study of a smallmolecule, a polypeptide or a nucleic acid (polynucleotide) encoding apolypeptide to reveal relevant chemical and biological information. Thisinformation generally includes one or more, but is not limited to, thefollowing: sequence information for protein and nucleic acid, primary,secondary, tertiary, and quarternary structure information, molecularweight, solubility in various solvents, enzymatic or other activity,isoelectric focusing point, binding affinity to other molecules, bindingpartners, stability, expression pattern, tissue distribution,subcellular localization, expression regulation, developmental roles,phenotypes of transgenic animals overexpressing or devoid of apolypeptide or nucleic acid, size of nucleic acid, and hybridizationproperty of nucleic acid. A variety of standard chemistry, cell andmolecular biology protocols and methodologies can be used, such as gelelectrophoresis, capillary electrophoresis, cloning, restriction enzymedigestion, expression profiling by hybridization, affinitychromatography, HPLC, isoelectric focusing, mass spectrometry, automatedsequencing, and the generation of transgenic animals, the details ofwhich can be found in many standard chemistry and molecular biologylaboratory manuals (see below). Techniques employing the hybridizationof nucleic acids may, for example, utilize arrayed libraries of nucleicacids, such as oligonucleotides, cDNA or others (See, for example, U.S.Pat. No. 5,837,832).

[0166] The term “chemically similar” is used to refer to chemicalcompounds with similar chemical structures and/or chemical properties.Similarity can be judged by comparison between two compounds of severalcharacteristics, such as electronic charge, steric size,stereochemistry, hydrogen bond donor/acceptor capability, and polarity(i.e., hydrophobicity/hydrophilicity). For example, chemically similaramino acids would have side chains which, judged by at least three,four, or preferably all five of these characteristics, are categorizedin the same way. For example, under physiological conditions, glycineand alanine are similar judged by all five characteristics, glycine andphenylalanine differ only judged by steric size, glycine and tyrosinediffer by steric size and hydrogen bond donor capability, and glycineand glutamic acid differ by steric size, charge, polarity, and hydrogenbond acceptor capability. For example, steroids are generally similar interms of conformation, polarity, stereochemistry, charge, steric size,etc., although some steroids (individually or as subclasses) may differslightly from “average” steroids (e.g., steroidal alkaloids aretypically charged under physiological conditions).

[0167] In certain embodiments, chemically similar small moleculecompounds share similar functional groups and/or ring systems and thusdisplay a combination of structural elements disposed in similarorientations or conformations, thereby defining a structural class ofcompounds which differ slightly, e.g., by substituents appended to thestructural core, or by slight variations in the structural core (such aschanges in ring size, heteroatom substitutions, homologation, etc.). Forexample, beta-lactam antibiotics all share a four-membered lactam ring,macrolide antibiotics have a macrocyclic lactone (e.g., 10 to 18members) substituted with multiple methyl and/or hydroxyl groups (someof the latter of which may be hydroxylated), peptides are chains ofalpha-amino acids linked by amide bonds, etc., and each such group ofcompounds comprises chemically similar members.

[0168] The term “derivative with minor modifications” with respect to aparent chemical compound, for example a small molecule, ligand, hybridligand, peptide or polypeptide, is used to refer to chemical compoundswhich are chemically similar to the parent chemical compound.Preferably, a derivative with minor modifications will have minorstructural modifications and hence may be considered as “structuralvariants” of the original compound. Generally, such minor structuralmodifications are made in order to obtain a compound with overallsimilar properties as compared to the parent compound, but with a changewith respect to a certain property of the parent compound that isdisadvantageous or unwanted. For example, a hydrophilic side chain maybe added to a certain chemical compound to increase its solubility,while retaining a desired biological activity as the side chain is addedsuch as not to interfere with the binding between the compound and itsbiological target.

[0169] A “chimeric polypeptide”, “fusion polypeptide” or “fusionprotein” is a fusion of a first amino acid sequence encoding a firstpolypeptide with a second amino acid sequence defining a domain (e.g.polypeptide portion) foreign to and not substantially homologous withany domain of the first polypeptide. Such second amino acid sequence maypresent a domain which is found (albeit in a different polypeptide) inan organism which also expresses the first polypeptide, or it may be an“interspecies”, “intergenic”, etc. fusion of polypeptide structuresexpressed by different kinds of organisms. At least one of the first andthe second polypeptides may also be partially or completely synthetic orrandom, i.e. not previously identified in any organism.

[0170] “To clone” as used herein, as will be apparent to skilledartisan, may be meant as obtaining exact copies of a givenpolynucleotide molecule using recombinant DNA technology. Furthermore,“to clone into” may be meant as inserting a given first polynucleotidesequence into a second polynucleotide sequence, preferably such that afunctional unit combining the functions of the first and the secondpolynucleotides results, for example, without limitation, apolynucleotide from which a fusion protein may be translationallyprovided, which fusion protein comprises amino acid sequences encoded bythe first and the second polynucleotide sequences. Details of molecularcloning can be found in a number of commonly used laboratory protocolbooks such as Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. bySambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:1989).

[0171] “To clone” as used herein, as will be apparent to skilledartisan, may be also meant as obtaining identical or nearly identicalpopulation of cells possessing a common given property, such as thepresence or absence of a fluorescent marker, or a positive or negativeselectable marker. The population of identical or nearly identical cellsobtained by cloning is also called a “clone.” Cell cloning methods arewell known in the art as described in many commonly available laboratorymanuals (see Current Protocols in Cell Biology, CD-ROM Edition, ed. byJuan S. Bonifacino, Jennifer Lippincott-Schwartz, Joe B. Harford, andKenneth M. Yamada, John Wiley & Sons, 1999).

[0172] “Complementation screen” as used herein means genetic screeningfor one or several genes or source DNA that can confer a certainspecified phenotype which will not exist without the presence of saidone or several genes or source DNA. It is usually done in vivo, byintroducing into cells lacking the specified phenotype a library ofsource DNA to be screened for, and identifying cells that have obtaineda source DNA and now exhibit the specified phenotype. Alternatively, itcould be done in vivo by randomly inactivating genes in the genome ofthe cell lacking the specified phenotype and identify cells that havelost the function of certain genes and exhibit the specified phenotype.However, a complementation screen can also be done in vitro in cell-freesystems, either by testing each candidate individually or as pools ofindividuals.

[0173] “Recovering a clone of the cell . . . under conditions wherein acell is selectable” as used herein is meant as selecting from apopulation of cells, a subpopulation or a single cell possessing a givenproperty such as the presence or absence of fluorescent markers, or thepresence or absence of positive or negative selectable markers, andobtaining a clone of each selected cell. The cells can be selected underconditions that will completely or nearly completely eliminate any cellthat does not have the desired property of the cells to be selected. Forexample, by growing cells in selective media, only cells possessing acertain desired property will survive. The surviving cells can be clonedusing standard cell and molecular biology protocols (see CurrentProtocols in Cell Biology, CD-ROM Edition, ed. by Juan S. Bonifacino,Jennifer Lippincott-Schwartz, Joe B. Harford, and Kenneth M. Yamada,John Wiley & Sons, 1999). Alternatively, cells possessing a desiredproperty can be selected from a population based on the observation of acertain discernable phenotype, such as the presence or absence offluorescent markers. The selected cells can then be cloned usingstandard cell and molecular biology protocols (see Current Protocols inCell Biology, CD-ROM Edition, ed. by Juan S. Bonifacino, JenniferLippincott-Schwartz, Joe B. Harford, and Kenneth M. Yamada, John Wiley &Sons, 1999).

[0174] The term “equivalent” is understood to include polypeptides ornucleotide sequences that are functionally equivalent or possess anequivalent activity as compared to a given polypeptide or nucleotidesequence. Equivalent nucleotide sequences will include sequences thatdiffer by one or more nucleotide substitutions, additions or deletions,such as allelic variants; and will, therefore, include sequences thatdiffer from the nucleotide sequence of a particular gene, due to thedegeneracy of the genetic code. Equivalent polypeptides will includepolypeptides that differ by one or more amino acid substitutions,additions or deletions, which amino acid substitutions, additions ordeletions leave the function and/or activity of the polypeptidesubstantially unaltered. A polypeptide equivalent to a given polypeptidecould e.g. be the polypeptide that performs the same function in anotherspecies. For example, murine ubiquitin herein is considered anequivalent of human ubiquitin.

[0175] “FK506 derivative” as used herein means a structural homolog ofnative FK506 in its broadest sense. It has been reported that FKBP, thenormal binding partner of FK506, can be modified to bind a FK506derivative in such a way that the mutated binding pocket can onlyaccommodate the FK506 derivative but not the wild type FK506 (Clacksonet al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:10437-42; and Yang etal., 2000, J. Med. Chem. 43:1135-42). It should be understood that theterm “FK506 derivative” covers at least this kind of FK506 derivativesin the context of binding complementary mutant FKBP. Furthermore, FK506derivatives can also be those structurally similar but not identicalcompounds which have essentially the same function as FK506.

[0176] “Reporter moiety” as used herein means a feature that can bedetected by certain means. For example, one routine assay for detectionis achieved by western blot using antibody specific for a proteinfeature. Alternatively, the reporter moiety or a reportermoiety-containing moiety may be capable of capable exhibiting anintended detectable function. Particularly, the function may besuppressed or inhibited before a certain event occurs (such as cleavageof the reporter moiety from the Cub-domain in a split ubiquitin system)and the suppression or inhibition may be abolished after such eventoccurs. For example, without limitation, a transcription reporter moietymay be rendered non-functional when it is attached to a Cub moiety thatis tethered to a membrane outside the nucleus of a target cell. It maybecome functional after cleavage of the reporter moiety from theCub-moiety when it can freely translocate to the nucleus to exert itstranscription activation/suppression function, which activity is in turndetectable by measuring the activity of a functionally linked reportergene.

[0177] As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding a polypeptide, including both exon and (optionally) intronsequences. The term “intron” refers to a DNA sequence present in a givengene which is not translated into protein and is generally found betweenexons.

[0178] The term “high affinity” as used herein means strong bindingaffinity between molecules with a dissociation Constance K_(D) of nogreater than 1 μM. In a preferred case, the K_(D) is less than 100 nM,10 nM, 1 nM, 100 pM, or even 10 pM or less. In a most preferredembodiment, the two molecules can be covalently linked (KD isessentially 0).

[0179] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules,with identity being a more strict comparison. Homology and identity caneach be determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity with anothersequence.

[0180] The term “interact” as used herein is meant to include allinteractions (e.g. biochemical, chemical, or biophysical interactions)between molecules, such as protein-protein, protein-nucleic acid,nucleic acid-nucleic acid, protein-small molecule, nucleic acid-smallmolecule or small molecule-small molecule interactions.

[0181] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject polypeptides preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the gene ingenomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

[0182] “Kit” as used herein means a collection of at least twocomponents constituting the kit. Together, the components constitute afunctional unit for a given purpose. Individual member components may bephysically packaged together or separately. For example, a kitcomprising an instruction for using the kit may or may not physicallyinclude the instruction with other individual member components.Instead, the instruction can be supplied as a separate member component,either in a paper form or an electronic form which may be supplied oncomputer readable memory device or downloaded from an internet website,or as recorded presentation.

[0183] “Instruction(s)” as used herein means documents describingrelevant materials or methodologies pertaining to a kit. These materialsmay include any combination of the following: background information,list of components and their availability information (purchaseinformation, etc.), brief or detailed protocols for using the kit,trouble-shooting, references, technical support, and any other relateddocuments. Instructions can be supplied with the kit or as a separatemember component, either as a paper form or an electronic form which maybe supplied on computer readable memory device or downloaded from aninternet website, or as recorded presentation. Instructions can compriseone or multiple documents, and are meant to include future updates.

[0184] “Library” as used herein generally means a multiplicity of membercomponents constituting the library which member components individuallydiffer with respect to at least one property, for example, a chemicalcompound library. Particularly, as will be apparent to skilled artisan,“library” means a plurality of nucleic acids/polynucleotides, preferablyin the form of vectors comprising functional elements (promoter,transcription factor binding sites, enhancer, etc.) necessary forexpression of polypeptides, either in vitro or in vivo, which arefunctionally linked to coding sequences for polypeptides. The vector canbe a plasmid or a viral-based vector suitable for expression inprokaryotes or eukaryotes or both, preferably for expression inmammalian cells. There should also be at least one, preferably multiplepairs of cloning sites for insertion of coding sequences into thelibrary, and for subsequent recovery or cloning of those codingsequences. The cloning sites can be restriction endonuclease recognitionsequences, or other recombination based recognition sequences such asloxP sequences for Cre recombinase, or the Gateway system (LifeTechnologies, Inc.) as described in U.S. Pat. No. 5,888,732, thecontents of which is incorporated by reference herein. Coding sequencesfor polypeptides can be cDNA, genomic DNA fragments, orrandom/semi-random polynucleotides. The methods for cDNA or genomic DNAlibrary construction are well-known in the art, which can be found in anumber of commonly used laboratory molecular biology manuals (seebelow).

[0185] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation, e.g., by agonizing or potentiating)and down-regulation (i.e. inhibition or suppression e.g., byantagonizing, decreasing or inhibiting) of an activity.

[0186] The term “mutation” or “mutated” as it refers to a gene ornucleic acid means an allelic or modified form of a gene or nucleicacid, which exhibits a different nucleotide sequence and/or an alteredphysical or chemical property as compared to the wild-type gene ornucleic acid. Generally, the mutation could alter the regulatorysequence of a gene without affecting the polypeptide sequence encoded bythe wild-type gene. But more commonly, a mutated gene or nucleic acidwill either completely lose the ability to encode a polypeptide (nullmutation) or encode a polypeptide with an altered property, including apolypeptide with reduced or enhanced biological activity, a polypeptidewith novel biological activity, or a polypeptide that interferes withthe function of the corresponding wild-type polypeptide. Alternatively,a mutation may take advantage of the degeneracy of the genetic code, byreplacing a triplet codon by a different triplet codon that neverthelessencodes the same amino acid as the wild-type triplet codon. Suchreplacement may, for example, lead to increased stability of the gene ornucleic acid under certain conditions. Furthermore, a mutation maycomprise a nucleotide change in a single position of the gene or nucleicacid, or in several positions, or deletions or additions of nucleotidesin one or several positions.

[0187] The term “reduced-associating mutant” as used herein means amutant polypeptide that exhibits reduced affinity for its normal bindingpartner. For example, a reduced-associating mutant of the ubiquitinN-terminus (Nux) is a polypeptide that exhibits reduced affinity for itsnormal binding partner—the C-terminal half of ubiquitin (Cub), to thepoint that it will show reduced association or not associate with awild-type Cub and form a “quasi-wild-type ubiquitin” without thesupplemented binding affinity between two polypeptides fused to Nux andCub, respectively. In a preferred embodiment of the invention, suchmutations in Nux are certain missense mutations introduced to either the3^(rd) or the 13^(th) amino acid residue of the wild-type ubiquitin.Different missense mutations at these positions may differentiallyaffect the affinity/association between Nux and Cub, thereby providingdifferent sensitivity of the assay as disclosed by the instantinvention. These missense point mutations can be routinely introducedinto cloned genes using standard molecular biology protocols, such assite-directed mutagenesis using PCR.

[0188] As used herein, the term “nucleic acid,” in its broadest sense,refers to polynucleotides such as deoxyribonucleic acid (DNA), and,where appropriate, ribonucleic acid (RNA). The term should also beunderstood to include, as equivalents, analogs of either RNA or DNA madefrom nucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or anti-sense) and double-strandedpolynucleotides.

[0189] Specifically, “nucleic acid(s)” may refer to polynucleotides thatcontain information required for transcription and/or translation ofpolypeptides encoded by the polynucleotides. These include, but are notlimited to, plasmids comprising transcription signals (e.g.transcription factor binding sites, promoters and/or enhancers)functionally linked to downstream coding sequences for polypeptides,genomic DNA fragments comprising transcription signals (e.g.transcription factor binding sites, promoters and/or enhancers)functionally linked to downstream coding sequences for polypeptides,cDNA fragments (linear or circular) comprising transcription signals(e.g. transcription factor binding sites, promoters and/or enhancers)functionally linked to downstream coding sequences for polypeptides, orRNA molecules comprising functional elements for translation either invitro or in vivo or both, which are functionally linked to sequencesencoding polypeptides. These polynucleotides should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or anti-sense) and double-strandedpolynucleotides. These polynucleotides can be in an isolated form, e.g.an isolated vector, or included into the episome or the genome of acell.

[0190] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0191] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a natural or recombinant geneproduct or fragment thereof which is not a nucleic acid.

[0192] The term “recombinant protein” refers to a polypeptide which isproduced by recombinant DNA techniques, wherein generally, DNA encodinga polypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the polypeptide encoded bysaid DNA. This polypeptide may be one that is naturally expressed by thehost cell, or it may be heterologous to the host cell, or the host cellmay have been engineered to have lost the capability to express thepolypeptide which is otherwise expressed in wild type forms of the hostcell. The polypeptide may also be a fusion polypeptide. Moreover, thephrase “derived from”, with respect to a recombinant gene, is meant toinclude within the meaning of “recombinant protein” those proteinshaving an amino acid sequence of a native polypeptide, or an amino acidsequence similar thereto which is generated by mutations, includingsubstitutions, deletions and truncation, of a naturally occurring formof the polypeptide.

[0193] “Small molecule” as used herein, is meant to refer to acomposition or compound, which has a molecular weight of less than about5 kD and most preferably less than about 4 kD. Small molecules can benucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates,lipids or other organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be potentially screened with methods of the invention by linkingsuch chemicals to a common ligand as used in the instant invention.

[0194] “Transcription” is a generic term used throughout thespecification to refer to a process of synthesizing RNA moleculesaccording to their corresponding DNA template sequences, which mayinclude initiation signals, enhancers, and promoters that induce orcontrol transcription of protein coding sequences with which they areoperably linked. “Transcriptional repressor,” as used herein, refers toany of various polypeptides of prokaryotic or eukaryotic origin, orwhich are synthetic artificial chimeric constructs, capable ofrepression either alone or in conjunction with other polypeptides andwhich repress transcription in either an active or a passive manner. Itwill also be understood that the transcription of a recombinant gene canbe under the control of transcriptional regulatory sequences which arethe same or which are different from those sequences which controltranscription of the naturally-occurring forms of the recombinant gene,or its components.

[0195] “Translation” as used herein is a generic term used to describethe synthesis of protein or polypeptide on a template, such as messengerRNA (mRNA). It is the making of a protein/polypeptide sequence bytranslating the genetic code of an mRNA molecule associated with aribosome. The whole process can be performed in vivo inside a cell usingprotein translation machinery of the cell, or be performed in vitrousing cell-free systems, such as reticulocyte lysates or any otherequivalents. The RNA template for translation may be separately providedeither directly as RNA or indirectly as the product of transcriptionfrom a provided DNA template, such as a plasmid.

[0196] “Translationally providing” means providing a polypeptide/proteinby way of translation. As defined above, translation is a process thatcan be done in vivo inside a cell using protein translation machinery ofthe cell, or be performed in vitro using cell-free systems, such asreticulocyte lysates or any other equivalents. The RNA template fortranslation may be separately provided either directly as RNA orindirectly as the product of transcription from a provided DNA template,such as a plasmid. The template DNA can be introduced into a host/targetcell by a variety of standard molecular biology procedures, such astransformation, transfection, mating or cell fusion, or can be providedto an in vitro translation reaction directly.

[0197] The terms “transfection” and “transformation” are usedinterchangeably herein to denominate the introduction of a nucleic acid,e.g., without limitation, via an expression vector, into a recipientcell.

[0198] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0199] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked may be referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of vectors which serveequivalent functions and which are known or become known in the artsubsequently hereto.

[0200] The “ubiquitins” are a class of proteins found in all eukaryoticcells. The ubiquitin polypeptide is characterized by a carboxy-terminalglycine residue that is activated by ATP to a high-energy thiol-esterintermediate in a reaction catalyzed by a ubiquitin-activating enzyme(E1). The activated ubiquitin is transferred to a substrate polypeptidevia an isopeptide bond between the activated carboxy-terminus ofubiquitin and the epsilon-amino group of (a) lysine residue(s) in theprotein substrate. This transfer requires the action of ubiquitinconjugating enzymes such as E2 and, in some instances, E3 activities.The ubiquitin modified substrate is thereby altered in biologicalfunction, and, in some instances, becomes a substrate for components ofthe ubiquitin-dependent proteolytic machinery which includes both UBPenzymes as well as proteolytic proteins which are subunits of theproteasome. As used herein, the term “ubiquitin” includes within itsscope all known as well as unidentified eukaryotic ubiquitin homologs ofvertebrate or invertebrate origin which can be classified as equivalentsof human ubiquitin. Examples of ubiquitin polypeptides as referred toherein include the human ubiquitin polypeptide which is encoded by thehuman ubiquitin encoding nucleic acid sequence (GenBank AccessionNumbers: U49869, X04803). Equivalent ubiquitin polypeptide encodingnucleotide sequences are understood to include those sequences thatdiffer by one or more nucleotide substitutions, additions or deletions,such as allelic variants; as well as sequences which differ from thenucleotide sequence encoding the human ubiquitin coding sequence due tothe degeneracy of the genetic code. Another example of a ubiquitinpolypeptide as referred to herein is murine ubiquitin which is encodedby the murine ubiquitin encoding nucleic acid sequence (GenBankAccession Number: X51730). It will be readily apparent to the personskilled in the art how to modify the methods and reagents provided bythe present invention to the use of ubiquitin polypeptides other thanhuman ubiquitin.

[0201] The term “ubiquitin-like protein” as used herein refers to agroup of naturally occurring proteins, not otherwise describable asubiquitin equivalents, but which nonetheless show strong amino acidhomology to human ubiquitin. As used herein this term includes thepolypeptides NEDD8, UBL1, NPVAC, and NPVOC. These “ubiquitin-likeproteins” are at least over 40% identical in sequence to the humanubiquitin polypeptide and contain a pair of carboxy-terminal glycineresidues which function in the activation and transfer of ubiquitin totarget substrates as described supra.

[0202] As used herein, the term “ubiquitin-related protein” as usedherein refers to a group of naturally occurring proteins, not otherwisedescribable as ubiquitin equivalents, but which nonetheless show somerelatively low degree (<40% identity) of amino acid homology to humanubiquitin. These “ubiquitin-related” proteins include human UbiquitinCross-Reactive Protein (UCRP, 36% identical to huUb, Accession No.P05161), FUBI (36% identical to huUb, GenBank Accession No. AA449261),and Sentrin/Sumo/Pic1 (20% identical to huUb, GenBank Accession No.U83117). The term “ubiquitin-related protein” as used herein furtherpertains to polypeptides possessing a carboxy-terminal pair of glycineresidues and which function as protein tags through activation of thecarboxy-terminal glycine residue and subsequent transfer to a proteinsubstrate.

[0203] The term “ubiquitin-homologous protein” as used herein refers toa group of naturally occurring proteins, not otherwise describable asubiquitin equivalents or ubiquitin-like or ubiquitin-related proteins,which appear functionally distinct from ubiquitin in their ability toact as protein tags, but which nonetheless show some degree of homologyto human ubiquitin (34-41% identity). These “ubiquitin-homologousproteins” include RAD23A (36% identical to huUb, SWISS-PROT. AccessionNo. P54725), RAD23B (34% identical to huUb, SWISS-PROT. Accession No.P54727), DSK2 (41% identical to huUb, GenBank Accession No. L40587), andGDX (41% identical to huUb, GenBank Accession No. J03589). The term“ubiquitin-homologous protein” as used herein is further meant tosignify a class of ubiquitin homologous polypeptides whose similarity toubiquitin does not include glycine residues in the carboxy-terminal andpenultimate residue positions. Said proteins appear functionallydistinct from ubiquitin, as well as ubiquitin-like and ubiquitin-relatedpolypeptides, in that, consistent with their lack of a conservedcarboxy-terminal glycine for use in an activation reaction, they havenot been demonstrated to serve as tags to other proteins by covalentlinkage.

[0204] The term “ubiquitin conjugation machinery” as used herein refersto a group of proteins which function in the ATP-dependent activationand transfer of ubiquitin to substrate proteins. The term thusencompasses: El enzymes, which transform the carboxy-terminal glycine ofubiquitin into a high energy thiol intermediate by an ATP-dependentreaction; E2 enzymes (the UBC genes), which transform the E1-S˜Ubiquitinactivated conjugate into an E2-S˜Ubiquitin intermediate which acts as aubiquitin donor to a substrate, another ubiquitin moiety (in apoly-ubiquitination reaction), or an E3; and the E3 enzymes (orubiquitin ligases) which facilitate the transfer of an activatedubiquitin molecule from an E2 to a substrate molecule or to anotherubiquitin moiety as part of a polyubiquitin chain. The term “ubiquitinconjugation machinery”, as used herein, is further meant to include allknown members of these groups as well as those members which have yet tobe discovered or characterized but which are sufficiently related byhomology to known ubiquitin conjugation enzymes so as to allow anindividual skilled in the art to readily identify it as a member of thisgroup. The term as used herein is meant to include novel ubiquitinactivating enzymes which have yet to be discovered as well as thosewhich function in the activation and conjugation of ubiquitin-like orubiquitin-related polypeptides to their substrates and topoly-ubiquitin-like or poly-ubiquitin-related protein chains.

[0205] The term “ubiquitin-dependent proteolytic machinery” as usedherein refers to proteolytic enzymes which function in the biochemicalpathways of ubiquitin, ubiquitin-like, and ubiquitin-related proteins.Such proteolytic enzymes include the ubiquitin C-terminal hydrolases,which hydrolyze the linkage between the carboxy-terminal glycine residueof ubiquitin and various adducts; UBPs, which hydrolyze theglycine76-lysine48 linkage between cross-linked ubiquitin moieties inpoly-ubiquitin conjugates; as well as other enzymes which function inthe removal of ubiquitin conjugates from ubiquitinated substrates(generally termed “deubiquitinating enzymes”). The aforementionedprotease activities function in the removal of ubiquitin units from aubiquitinated substrate following or during uibiquitin-dependentdegradation as well as in certain proofreading functions in which freeubiquitin polypeptides are removed from incorrectly ubiquitinatedproteins. The term “ubiquitin-dependent proteolytic machinery” as usedherein is also meant to encompass the proteolytic subunits of theproteasome (including human proteasome subunits C2, C3, C5, C8, and C9).The term “ubiquitin-dependent proteolytic machinery” as used herein thusencompasses two classes of proteases: the deubiquitinating enzymes andthe proteasome subunits. The protease functions of the proteasomesubunits are not known to occur outside the context of the assembledproteasome, however independent functioning of these polypeptides hasnot been excluded.

[0206] The term “kinase” as used herein refers to an enzyme thattransfers a phosphate group from a nucleoside triphosphate to anothermolecule. Preferably, the kinase is selected from the following list:AMP-PK (AMP-activated protein kinase, acetyl-CoA carboxylase kinase-3,HMG-CoA reductase kinase, hormone-sensitive lipase kinase), ACK2(acetyl-CoA carboxylase kinase-2), AFK (actin-fragmin kinase), APL-A1(Aplysia Californica cAMP-dependent PK 1), APL-A2 (Aplysia CalifornicacAMP-dependent PK 2), CAK (Cdk-activating kinase), CAMII (=CaM-II),beta-ARK1 (beta-adrenergic receptor kinase 1=GRK2), beta-ARK2(beta-adrenergic receptor kinase 2=GRK3), c-Ab1 (cellular Ab1), c-Raf(cellular Raf), c-Src (cellular Src), Cdk (cyclin dependent kinase),cdc2 (cell division cycle protein kinase), CK (casein kinase), CK-I orCKI (casein kinase I), CK-II or CKII (casein kinase II), CTD kinase((RNA polymerase II) carboxy-terminal domain kinase), CaM-I(calmodulin-dependent protein kinase I), CaM-II (calmodulin-dependentprotein kinase II, calmodulin-dependent multiprotein kinase, CaM-MPK),CaM-III (calmodulin-dependent protein kinase III, EF-2 kinase), DNA-PK(DNA-dependent protein kinase), ds-DNA kinase (double-strandedDNA-activated protein kinase), ds-RNA kinase (double strandedRNA-activated protein kinase, p68 kinase), EGF-R or EGFR (epidermalgrowth factor receptor), ERK (extracellular signal regulatedkinase=MAPK), ERT PK (growth factor-regulated kinase), FAK (focaladhesion kinase), GRK1 (G protein-coupled receptor kinase 1=RK), GRK2 (Gprotein-coupled receptor kinase 2=beta-ARK1), GRK3 (G protein-coupledreceptor kinase 3=beta-ARK2), GRK4 (G protein-coupled receptor kinase4), GRK5 (G protein-coupled receptor kinase 5), GRK6 (G protein-coupledreceptor kinase 5), GSK1 (glycogen synthase kinase 1=PKA), GSK2(glycogen synthase kinase 2=PHK), GSK3 (glycogen synthase kinase 3),GSK4 (glycogen synthase kinase 4), GSK5 (glycogen synthase kinase5=CKII), HI-HK (growth-associated HI histone kinase (MPF), cdc2+/CDC28protein kinase) H4-PK (histone-H4-specific, protease activated proteinkinase), H4-PK-I (histone H4 kinase I), H4-PK-II (histone H4 kinase II),HCR (heme-controlled repressor, heme-regulated eIF-2-alpha kinase), HKII(histone kinase II), INS—R or INSR (insulin receptor), Jak1 (Janusprotein-tyrosine kinase 1), Jak2 (Janus protein-tyrosine kinase 2),LCK/FYN (LYMPHOCYTE-SPECIFIC PROTEIN TYROSINE KINASE P56LCK), MAPK(mitogen-activated protein kinase (MAP kinase)=ERK), MAPKAPK-1 (MAPkinase-activated protein kinase 1=S6K-II), MAPKAPK-2 (MAPkinase-activated protein kinase 2), MEK (MAP, Erk kinase, MAP kinasekinase), MFPK (multifunctional protein kinase), MHCK (myosin heavy chainkinase), MLCK (myosin light chain kinase), p135tyk2 (135 kD tyk2tyrosine-protein kinase), p34cdc2 (34 kD cell division cycle proteinkinase), p42cdc2 (42 kD cell division cycle protein kinase), p42mapk (42kD MAP kinase isoform), p44 mpk (44 kD meiosis-activated myelin basicprotein kinase=ERK1), p60-src (tyrosin-protein kinase src), p74raf-1 (74kDa protein kinase Raf isoform), PDGF-R or PDGFR (platelet-derivedgrowth factor receptor), PHK (phosphorylase kinase), PI-3 kinase(phosphatidylinositol 3′ kinase), PKA (cAMP-dependent protein kinase,protein kinase A), PKC (protein kinase C), PKG (cGMP-dependent proteinkinase), PRK1 (lipid-activated PKC-related kinase), Raf (protein kinaseRat), RK (rhodopsin kinase=GRK1), RS kinase (nuclear envelope-boundprotein kinase), S6K (S6 kinase), S6K-II (S6-kinase 2=MAPKAPK-1), v-Src(viral Src).

[0207] The term to “bind to or inhibit a kinase” refers to the abilityof certain compounds to bind to kinases with high affinity, and thefurther property of certain compounds to lower the activity of a kinase.The “or” therein is not meant exclusive, i.e. a compound may both bindto a kinase and inhibit it, or it may only bind, or it may only inhibitsuch kinase, as the case may be.

[0208] 3. In-vitro uses of the Molecules of the Present Invention

[0209] The hybrid ligands of the present invention may be usedadvantageously in in vitro pull-down experiments. For example, they maybe used in a method to identify a ligand which binds to a givenpolypeptide P2. To this purpose, one, or a library of, hybrid ligand(s)of the general structure R1-Y-R2 as further substantiated above could besynthesized by one of the methods given below, wherein R1 is chosen tobind specifically to a known polypeptide P1, and R2 is a one, or acollection of, candidate ligand(s) to be screened for binding to P2. P1may then be immobilized on a matrix, and the matrix incubated withsolutions containing the one, or a library of, hybrid ligand(s) R1-Y-R2,preferably in a manner that allows a resolution of different members ofthe library if several are to be employed. Subsequently, the matrix isincubated with a solution containing P2, wherein P2 may be labelled foreasy detection, such that binding of P2 to a ligand R2 with highaffinity for P2 may occur. Finally, P2 immobilized to the matrix throughbinding to a ligand R2 with high affinity for P2 is detected, and thecorresponding hybrid ligand may be isolated for further analysis.

[0210] In such embodiment, the matrix may, for example, be chosen to bea collection of beads, a particulate resin, a gel, a porous membrane ora solid surface, for example a glass surface. Usually, theimmobilization of P1 on the matrix surface will be performed in batchmode. The method of choice for immobilization of P1 on the surface willdepend on P1 and the choice of matrix. Preferably, a covalent bond iscreated between P1 and the matrix surface; where the binding between P1and the matrix is non-covalent, the matrix should be chosen such as tomaximize the affinity of P1 for binding to the matrix. For example, thesurface may have been pre-treated such that it displays sulfhydrylgroups, which may be reacted with cystein groups on P1 to form disulfidebridges. Another convenient method is the use of matrices withimidazolyl-carbamate groups on their surface (e.g. Reacti-gel, PierceBiotechnology, Rockford, Ill., USA, Cat. # 20259). Many other methods toimmobilize proteins on surfaces are known to the skilled person, and maybe applied where appropriate; see, for example, Hermanson, G. T.,Bioconjugate Techniques (1996), Academic Press, San Diego, Calif., USA.

[0211] Where the matrix is chosen to be a collection of beads or aparticulate resin, after the immoblization of P1 in batch mode the batchwill preferably be divided up into fractions, and each fraction may thenbe treated with a different hybrid linker R1-Y-R2, differing only in R2,for example in different wells of a microtiter plate. When the hybridligands have bound to the matrix via the association of R1 to P1, asample containg the polypeptide P2 is added and allowed to bind. Thismay be performed keeping the beads or resin particles with the differenthybrid ligands separated. Alternatively, if a suitable separationtechnique is available, the beads or resin particles may be pooledbefore exposure to P2; subsequent separation can, for example, beperformed by using a fluorescently labelled P2, and, for example,sorting fluorescent from non-fluorescent beads on a fluorescenceassisted sorter.

[0212] Where a gel, membrane or solid surface is chosen as a matrix,after preferably uniform immobilization of P1 on the matrix, the libraryof hybrid ligands, if several are employed, is preferably distributed onthe matrix in a spatially resolved format, i.e. in a dotted grid whereat every location on the matrix, there is only one hybrid ligand, oronly one pool of hybrid ligands. Preferably, such a grid is addressable,and/or the identity of the hybrid ligands present at a given location onthe matrix is known. The matrix is then incubated with a solutioncontaining the polypeptide P2, such that P2 may bind to any R2 withaffinity for P2. After washing away unbound P2, bound P2 may be detectedin situ or after dissociation from the matrix, for example by detectingthe presence of a fluorescent or enzymatically active tag present on P2,or by reacting the matrix with a labelled antibody with affinity for P2,or by methods such as surface plasmon resonance or mass spectrometry, orby other methods to detect the presence of a polypeptide well known tothe skilled person.

[0213] Alternatively, the hybrid ligands of the invention may be used ina method to identify a polypeptide P2 which binds to a given smallmolecule ligand R2. Such a method will be almost identical to themethods described in the preceding paragraphs, except that only onehybrid ligand R1-Y-R2 is synthesized and incubated with the matrixpretreated with P1, and the matrix with the immobilized complexesP1::R1-Y-R2 is incubated with a sample containing one or a plurality ofcandidate binding partner/interactor polypeptide(s) P2, either in poolsor separately if several are employed. Such a sample could, for example,be a cell extract. After eluting unbound P2, bound P2 may be detected asdescribed in the previous paragraph. In order to identify a P2 that isdetected as binding to the immobilized P1::R1-Y-R2 complex, it may bedissociated from the matrix and isolated by any known method, forexample by competitive displacement using isolated R2 or anotherpolypeptide known to bind strongly to R2, or denaturation of peptides bydrastic pH change or addition of other denaturing agents such as 6 Mguanidine-HCl, 2 M urea, or 10 mM DTT. The identification of P2 isconveniently carried out by mass spectrometry, either of isolated P2, orafter degradation of the polypeptides on the matrix, for example byincubating the matrix with a solution containing trypsin. Other methodsto identify polypeptides are known to the skilled person and may beequally used, such as amino acid sequencing WO 93/08278, WO 98/37186, WO01/14539 and WO 02/22826 describe biological systems for theinvestigation of protein-protein interactions which may be used in sucha manner with the hybrid ligands and methods of the present invention.These documents provide nucleic acids and nucleic acid libraries toidentify polypeptides that bind to a molecule of interest. These nucleicacids encode fusion polypeptides, comprising the polypeptide to betested for interaction with the molecule of interest, and a seconddomain which is capable to bind to a certain nucleic acid sequence motifwith high affinity, or even covalently as described in WO 98/37186, WO01/14539 and WO 02/22826. The vector for the expression of the fusionpolypeptides is designed to comprise the nucleic acid sequence motifwhich the nucleic acid binding domain of the fusion polypeptides bindsto. Therefore, when this vector is introduced into cells and thepolypeptide is expressed, it will bind to the vector which encodesitself, forming a polypeptide-nucleic acid-complex. These complexes maybe isolated from the cells used for their expression, and applied to ascreening assay which identifies polypeptides that bind to a hybridligand of the invention.

[0214] To this end, at least one hybrid ligand R1-Y-R2 as detailed aboveis synthesized, as well as a library of vectors encoding DNA-bindingfusion polypeptides. The construction of variegated libraries isdetailed below, further guidance is given, for example, in WO 93/08278,WO 98/37186, WO 01/14539 and WO 02/22826, which are incorporated hereinby reference. These vectors are introduced into suitable host cells,preferably mammalian cells if post-translational modifications ofpolypeptides need be incorporated, and the cells propagated to thedesired quantity.

[0215] Subsequently, the host cells may be lysed and the polypeptide-DNAcomplexes separated from cellular debris; optionally, furtherpurification steps may be performed, such as centrifugation or gelfiltration. The library of polypeptide-DNA complexes may then beincubated in solution with the hybrid ligand(s) directly andsubsequently passed over a matrix with immobilized P1 on its surface asdescribed above, or incubated with a matrix which has been pre-treatedwith the hybrid ligand. Alternatively, host cells may be treated withthe hybrid ligand, then lysed, and the lysates incubated with or passedover the P1-bearing matrix, optionally after one or more purificationsteps.

[0216] After washing away unbound complexes, those complexes that didestablish binding to the hybrid ligand may be recovered from the matrixas described above, and the DNA isolated for sequencing to identify P2.Alternatively, the binding of the DNA to the surface via the DNA-bindingdomain of P2 may be abolished, for example by denaturing the DNA, andonly the DNA encoding P2 may be isolated and sequenced. For example, theplasmid encoding P2 may contain restriction sites flanking the bindingsite of the DNA binding domain fused to P2, allowing the elution andisolation of a linear DNA fragment after treatment of the complexes withrestriction enzymes. Alternatively, a protease may be used to cleave theP2 fusion protein, or the P2 fusion protein may be denatured, forexample by treatment with SDS or mercaptoethanol, and the DNA elutedfrom the matrix.

[0217] DNA isolated from the matrix will usually be amplified by PCRbefore sequencing. However, the part of the plasmid encoding P2 may bedirectly amplified on the matrix using appropriate primers, and the PCRproduct used for sequencing. Sequencing will usually be carried out bywell established methods, such as Dideoxy-mediated sequencing usingT7-DNA-Polymerase or Klenow-Polymerase or Taq-Polymerase,Cycle-Sequencing using Taq-Polymerase or Chemical Sequencing(Maxam-Gilbert sequencing), but other methods may be used, even thosedeveloped subsequently hereto.

[0218] Either way, this method directly yields genetic information onthe identity of polypeptides which bind R2, which greatly facilitatesthe identification of P2. DNA sequencing is cheaper, faster and lesscumbersome when trying to obtain polypeptide sequence information thanmost methods that employ the polypeptide itself. Example 4 hereinexemplifies the use of such method as contemplated herein, usingnon-covalent bonding between the gene product of lacI, the lacrepressor, to the lac operon DNA sequence on the plasmid. However, othercombinations of DNA motif and DNA binding polypeptide may be used andare within the scope of the present invention, including combinationswherein the binding between the DNA binding domain and the DNA motif iscovalent, and specifically those described in WO 98/37186, WO 01/14539and WO 02/22826. More specifically, the DNA binding protein may be anucleic acid modification (NAM) protein, which covalently attaches to anenzyme attachment sequence (EAS). Suitable NAM proteins include, but arenot limited to, Rep proteins, specifically Rep68 and Rep78 proteins, ofadeno-associated viruses, NS1 and H-1 proteins of parvovirus,bacteriophage phi-29 terminal proteins and the 55 Kd adenovirusproteins, and fragments and derivatives thereof which retain therespective DNA binding activity, although homologues of these proteinsfrom other viruses may also be employed. Suitable EAS for use with theseDNA binding proteins may be taken from the literature, e.g. WO 98/37186,WO 01/14539 and WO 02/22826.

[0219] In the above methods, R1 preferably represents a first ligandselected from: steroid, retinoic acid, beta-lactam antibiotic,cannabinoid, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, novobiocin, maltose, glutathione, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone,2,4-diaminopteridine derivative or cyclosporin, or a derivative thereofwith minor modifications.

[0220] P1 may be chosen to be a fusion polypeptide, comprising at leasttwo domains which are not found in combination in nature.

[0221] Such fusion polypeptide P1 may comprise one domain chosen fromthe group consisting of: β-lactamase, a steroid receptor, retinoic acidreceptor, cannabinoid receptor, FKB 12, Tet-R, DHER, GyrB, maltosebinding protein, glutathione-S-transferase, vitamin D receptor,glucocorticoid receptor, estrogen receptor, progesterone receptor,testosterone receptor, or a fragment thereof retaining the bindingcapacity to its respective ligand, and a second domain comprising a tagwhich allows the immobilization of said fusion protein on a matrix. Suchtag may be chosen from the group consisting of: strep tag, FLAG tag,his₆-tag, CBD tag, E-tag, GFP tag, GST tag, haemagglutinin tag, Myc tag,T7 tag, Tag 100, V5 tag, Calmodulin binding peptide tag, S tag,Intein/chitin binding domain tag, Xpress tag, thioredoxin tag or VSVtag.

[0222] 4. Transcriptional and Other Reporter Systems

[0223] According to another embodiment of the invention, a reportersystem is used to detect the proximity of two polypeptides P1 and P2 (asdefined above) when a small molecule compound is present so that eitherthe small molecule compound or one of the polypeptides can be identifiedand further characterized.

[0224] The following sections will describe a variety of reportersystems that can be used in the invention. It will be readily apparentto the skilled artisan that the immediate invention may also be used inconjunction with other reporter systems, even those that are developedin the future.

[0225] 4.1 Split Ubiquitin Reporter Systems

[0226] In part, the invention is based upon the finding that eventransient interactions can be detected using a novel split ubiquitinbased polypeptide association selection method. The split ubiquitinmethod has been used to demonstrate, for example, the association ofSec63p with various other yeast membrane proteins which traffic throughthe endoplasmic reticulum (ER) and the Golgi apparatus or are targetedto the plasma membrane.

[0227] The invention is understood to encompass modifications andextensions of the above described examples as follows.

[0228] The invention provides a fusion protein comprising P1-Cub-Z-RMpolypeptide, where P1 is a first polypeptide, Cub is a C-terminalsub-domain of ubiquitin, Z is an amino acid residue and RM is a reportermoiety wherein the fusion protein is cleavable by a ubiquitin-specificprotease in the presence of an interacting wild-type or mutant form ofthe Nub sub-domain of ubiquitin fused to a second polypeptide P2 (P2-Nuxfusion) and results in the release of the reporter moiety. Depending onthe identity of residue Z, the released RM may be stable if Z is Met andunstable if Z is a non-methionine amino-terminal amino acid, thus theactivity of said reporter moiety can be changed before and/or after saidrelease. The affinity between the Cub and Nub may be modulated byintroducing point mutations (for example, at residues 3 or 13 or bothpositions) into Nub so that Cub and Nub (or its derivative mutant forms“Nux”) can not interact with each other without the presence of otherstabilizing forces such as the one provided by interaction between P1and P2, in this case indirectly, through a compound ligand. It should beunderstood that due to the symmetric nature of the system, thedesignation of P1/P2 and R1/R2 is arbitrary. The reporter moiety ofthese fusion proteins may be a variety of proteins including, but notlimited to: a negative selectable marker, a positive selectable marker,a metabolic marker, a transcription factor, and a fluorescent marker. Inpreferred applications, the reporter is a selectable marker which iscapable of both positive and negative selection such as URA3, HygTk,Tkneo, TKBSD, PACTK, HygCoda, Codaneo, CodaBSD, and PACCoda. Otherreporters include LYS2, HIS3 and mammalian GPT. The reporter moiety mayalso be a fluorescent marker, a transcription factor, e.g. PLV (Stagljaret al., PNAS, 1998, 95:5187-92), or DHFR.

[0229] The invention uses peptide libraries expressed as fusionproteins. Such peptide libraries may be synthetic, natural, random,biased-random, constrained, non-constrained and combinatorial peptidelibraries. In certain instances, the peptide libraries are provided byexpression of nucleic acid construct(s) encoding the polypeptides. TheDNA libraries may be cDNA, random, biased-random, synthetic, genomic oroligonucleotide nucleic acid construct(s) encoding polypeptides.

[0230] The invention further provides a method of detecting the bindingof a chemical compound to a protein comprising: providing a firstprotein as a first polypeptide fusion comprising the structureP1-Cub-Z-RM polypeptide, where P1 is a first polypeptide, Cub is aC-terminal sub-domain of ubiquitin, Z is an amino acid residue and RM isa reporter moiety; providing a second fusion protein as a secondpolypeptide fusion comprising the structure P2-Nux where P2 is a secondpolypeptide and Nux is a wild-type or mutant form of an amino-terminalsub-domain of ubiquitin; providing a chemical compound of the generalformula R1-Y-R2 wherein R1 is a known ligand for P1, R2 is a potentialligand for P2, and Y is a linker sequence; allowing the chemicalcompound to come into close proximity with the first polypeptide fusionand the second polypeptide fusion under conditions wherein if R2interacts with P2, and cleavage of the first fusion protein results inrelease of the reporter moiety having the amino-terminal amino acidresidue Z; providing conditions that allow the detection of activity ofthe reporter moiety wherein the presence or absence of a detectablesignal from the reporter moiety indicates that the chemical compound R2binds P2. It should be understood that due to the symmetric nature ofthe system, the designation of P1/P2 and R1/R2 is arbitrary and eitherP1 or P2 can be fused to Cub-Z-RM. Similarly, in the PI-Nux fusionprotein, it should be understood that, unless specifically specified,P1-Nux refers to either of the two possible configurations of the fusionprotein, namely P1-Nux (N-terminal fusion) or Nux-P 1 (C-terminalfusion). In addition, P1-Cub-Z-RM is understood to encompass allpossible configurations of the fusion protein as long as it is in anorder wherein Cub-Z is closer to the N-terminus of the fusion proteinthan RM (for example, P1-Cub-Z-RM, Cub-Z-P1-RM, and Cub-Z-RM-PI are allpossible configurations).

[0231] In a preferred embodiment, P1 and R1 are known to interact witheach other while either the ligand binding to known protein P2 orprotein P2 binding to known ligand R2 can be identified and furthercharacterized.

[0232] This method of the invention may be performed in an in vitro oran in vivo format. The in vivo formats may utilize a host cell such as aeukaryotic cell. Suitable eukaryotic cells include mammalian cellsincluding human, mouse, rat, and hamster cells; vertebrate cellsincluding zebra fish cells; invertebrate cells including Drosophila andnematode cells; and fungal cells including S. pombe and S. cerevisiaecells. In preferred in vivo embodiments of the method of the invention,the reporter moiety is a positive selectable marker. The reporter mayalso be a negative selectable marker. The marker may be a metabolicmarker, a transcription factor, both a positive and negative selectablemarker, a fluorescent marker, a transcription factor, or DHFR. Themethod provides for the use of various amino acid residues to beengineered to the presumptive amino terminus of the reporter orselectable marker protein. In one embodiment, this amino acid isarginine, however it may also be an other non-methionine amino acid—e.g.lysine or histidine. In another embodiments, Z can be methionine orother stable amino acids in a given environment (see below).

[0233] The method of the invention uses first and/or secondpolypeptides, P1 and/or P2 which may be supplied as synthetic, natural,random, biased-random, constrained, non-constrained and combinatorialpeptide libraries. These libraries may be provided by expression ofnucleic acid construct(s) encoding said first and/or secondpolypeptides. The method of the invention also uses a fusion proteincomprising P2 and Nux, wherein the Nux is fused to the N-terminus of thesecond polypeptide P2 or to the C-terminus of the second polypeptide P2.

[0234] The method of the invention provides chemical compound R1-Y-R2,which may be supplied as synthetic or natural or other chemical compoundlibraries.

[0235] 4.1.1 Selectable Markers

[0236] The principle set up of the current split ubiquitin proteinsensor technology employs two yeast/E. coli shuttle vectors coding forthe “bait-Cub-Reporter” and the “Nub-prey” fusion proteins, where Nuband Cub stand for the respective N- and C-terminal halves of theubiquitin monomer (Johnsson & Varshavsky, 1994, Proc. Natl. Acad. Sci.U.S.A. 91:10340-10344).

[0237] Upon interaction between bait and prey through a chemicalcompound R1-Y-R2, the ubiquitin halves are brought into close contactand re-associate to form a unit that is sufficiently well recognized byUBPs (ubiquitin-specific-proteases). This recognition event leads toproteolytic cleavage and subsequent release of the C-terminally fusedreporter.

[0238] In a typical 3-hybrid approach re-association of the ubiquitinhalves with subsequent release of the reporter would rely on a smallmolecule-protein interaction, rather than protein-protein interaction.The bait construct would employ a “receptor-Cub-reporter” (P1-Cub-RM)fusion. Similarly to the split ubiquitin protein sensor technology, the“Receptor-Cub-reporter” and the Nub-prey constructs are expressed from 2separate shuttle vectors. The small molecule to be investigated is fusedto a common functional group that binds to the “receptor”. The receptormay be DHFR (dehydrofolate reductase). Here, DHFR functions as receptorfor the common functional group methotrexate (Mtx). Mtx or itsderivatives with a similar functional group (such as2,4-diaminopteridine) will be fused to various small molecules withnumerous different linker molecules. The small molecule itself will beanalyzed for its interaction with proteins present in a Nub-preylibrary. Interaction of the compound with a prey will lead to bridgingof R-Cub-DHFR::Mtx-small molecule::prey-Nub, thereby bringing Cub andNub (or Nux) into close contact, leading to release of the reportermoiety RM.

[0239] The reporter moiety may trigger any sort of detectable change,i.e. may rely on detection of proteolytic splice products by gelelectrophoresis and/or western blot analysis, enzymatic or fluorescencereadout, nutritional complementation, or other forms of transcriptionalreadout.

[0240] The reporter moiety may be a transcription factor tethered to acellular membrane preventing entry into the nucleus and transcriptionalactivation. Only upon re-association of the ubiquitin halves aftercompound-protein interaction, the reporter moiety will be released andtranslocate into the nucleus where transcription of a reporter gene maybe activated. Reporter genes may be enzymes, fluorescent markers ornutritional markers (e.g. lacZ, green fluorescent protein GFP/ yeastcodon optimized red fluorescent protein yRFP, HISIURA) (Stagljar et al.(1998) Proc. Natl. Acad. Sci. U.S.A., 95: 5187-92).

[0241] The invention uses negative selectable marker genes or“selectable reporters” which can be used in a eukaryotic host cell,preferably a yeast or a mammalian cell, or a prokaryotic cell, and whichcan be selected against under appropriate conditions. In preferredembodiments, the selectable reporter is provided as a fusion polypeptidewith a carboxy- or C-terminal sub-domain of ubiquitin (or Cub) and isaltered so as to encode a non-methionine amino acid residue at thejunction with the Cub. The non-methionine amino acid residue ispreferably an amino acid which is recognized by the N-end rule ubiquitinprotease system (e.g. an arginine, lysine histidine, phenylalanine,tryptophan, tyrosine, leucine or isoleucine residue) and which, whenpresent at the amino-terminal end of the negative selectable marker,targets the negative selectable marker for rapid proteolyticdegradation.

[0242] A preferred example of a selectable marker gene for use in yeastis the URA3 gene which can be both selected for (positive selection) bygrowing ura3 auxotrophic yeast strains in the absence of uracil, andselected against (negative selection) by growing cells on mediacontaining 5-fluoroorotic acid (5-FOA) (see Boeke, et al. (1987) MethodsEnzymol 154: 164-75). The concentration of 5-FOA can be optimized bytitration so as to maximally select for cells in which the URA3 reporteris, for example, inactivated by proteolytic degradation to somepreferred extent. For example, relatively high concentrations of 5-FOAcan be used which allow only cells expressing very low steady-statelevels of URA3 reporter to survive. Such cells will correspond to thosein which the first and second ubiquitin sub-domain fusion proteins havea relatively high affinity for one another, resulting in efficientreassembly of the Nub and Cub fragments and a correspondingly efficientrelease of the Z-URA3 labilized marker. In contrast, lowerconcentrations of 5-FOA can be used to select for protein bindingpartners with relatively weak affinities for one another. In addition,proline can be used in the media as a nitrogen source to make the cellshypersensitive to the toxic affects of the 5-FOA (McCusker & Davis(1991) Yeast 7: 607-8). Accordingly, proline concentrations, as well as5-FOA concentrations can be titrated so as to obtain an optimalselection for URA3 reporter deficient cells. Therefore the use of URA3as a negative selectable marker allows a broad range of selectivestringencies which can be adapted to minimize false positive backgroundnoise and/or to optimize selection for high affinity bindinginteractions. Other negative selectable markers which operate in yeastand which can be adapted to the method of the invention are includedwithin the scope of the invention.

[0243] Numerous selectable markers which operate in mammalian cells areknown in the art and can be adapted to the method of the invention so asto allow direct negative selection of interacting proteins in mammaliancells. Examples of mammalian negative selectable markers includeThymidine kinase (Tk) (Wigler et al. (1977) Cell 11: 223-32; Borrelli etal. (1988) Proc. Natl. Acad. Sci. USA 85: 7572-76) of the Herpes Simplexvirus, the human gene for hypoxanthine phosphoriboxyl transferase (HPRT)(Lester et al. (1980) Somatic Cell Genet. 6: 241-59; Albertini et al.(1985) Nature 316: 369-71) and Cytidine deaminase (codA) from E. coli(Mullen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 33-37; Wei andHuber (1996) J. Biol. Chem. 271: 3812-16). For example: the Tk gene canbe selected against using Gancyclovir (GANC) (e.g. using a 1 μMconcentration) and codA gene can be selected against using 5-FluorCytidin (5-FIC) (e.g. using a 0.1-1.0 mg/ml concentration). In addition,certain chimeric selectable markers have been reported (Karreman (1998)Gene 218: 57-61) in which a functional mammalian negative selectablemarker is fused to a functional mammalian positive selectable markersuch as Hygromycin resistance (Hyg^(R), neomycin resistance (neo^(R)),puromycin resistance (PAC^(R)) or Blasticidin S resistance (BlaS^(R)).These produce various Tk-based positive/negative selectable markers formammalian cells such as HygTk, Tkneo, TKBSD, and PACTK, as well asvarious codA-based positive/negative selectable markers for mammaliancells such as HygCoda, Codaneo, CodaBSD, and PACCoda. Tk-neo reporterswhich incorporate luciferase, green fluorescent protein and/orbeta-galactosidase have also been recently reported (Strathdee et al.(2000) BioTechniques 28: 210-14). These vectors have the advantage ofallowing ready screening of the “positive” marker/reporter byfluorescent and/or immunofluorescent microscopy. The use of suchpositive/negative selectable markers affords the advantages mentionedabove for URA3 as a reporter in yeast, inasmuch as they allow mammaliancells to be assessed by both positive and negative selection methods forthe expression and relative steady-state level of the reporter fusion.Other advantages of these mammalian reporter and selectable markerconstructs will be apparent to the skilled artisan.

[0244] 4.1.2 Components of N-End Rule Proteolytic Pathway

[0245] The “N-end rule” system for proteolytic degradation is aparticular branch of the ubiquitin-mediated proteolytic pathway presentin eukaryotic cells (Bachmair et al. (1986) Science 234: 179-86). Thissystem operates to degrade a cellular polypeptide at a rate dependentupon the amino-terminal amino acid residue of that polypeptide. Proteintranslation ordinarily initiates with an ATG methionine codon and somost polypeptides have an amino-terminal methionine residue and aretypically relatively stable in vivo. For example, in the yeast S.cerevisiae, a beta-galactosidase polypeptide with a methionine aminoterminus has a half-life of >20 hours (Varshavsky (1992) Cell 725-35).Under certain circumstances, however, polypeptides possessing anon-methionine amino-terminal residue can be created. For example, whenan endoprotease hydrolyzes and thus cleaves a unique polypeptide bond(A-B) internal to a polypeptide, it results in the release of twoseparate polypeptides—one of which possesses an amino-terminal aminoacid, Z, which may not be methionine. For example, the endoproteaseubiquitin-specific protease, which is a preferred component of thepresent invention, will cleave a polypeptide bond carboxy-terminal tothe final glycine residue (codon 76), regardless of what the next codonis. In the normal function of the cell, this-specific protease serves tocleave a polyubiquitin precursor into individual ubiquitin units.However it can also be used to generate a target polypeptide withvirtually any amino-terminal residue by merely fusing the targetpolypeptide in-frame to a codon corresponding to the desiredamino-terminal amino acid (Z), which codon, in turn, is fused downstreamof ubiquitin (typically contiguous with ubiquitin Gly codon 76). Theresulting target gene chimera construct, has the general formulaUbiquitin-Z-Target. Preferred target constructs further comprise anepitope tag (Ep) so that the resulting target gene chimera construct hasthe general formula Ubiquitin-Z-Ep-target, which results in the eventualproduction of a polypeptide of the general formula Z-Ep-Target.Constitutively active ubiquitin-specific protease activities present ineucaryotic cells will result in the endoproteolytic processing of theUbiquitin-Z-Target polypeptide into ubiquitin and Z-Target entities. TheZ-Target polypeptide is further acted upon by the components of theN-end rule system as described below. If the Target polypeptide is anegative selection marker (NSM) and if Z is an amino acid residue (suchas arg) which potentiates rapid degradation by the N-end rule system,then cells expressing intact Ubiquitin-Z-NSM can be selected againstwhile cells in which the fusion is clipped into a relatively labileZ-NSM polypeptide can be selected for.

[0246] It has been determined, with reasonable reliability, the relativeeffect of a given amino-terminal residue, Z, upon target polypeptidestability. For example, when all 20 possible amino-terminal amino acidresidues were tested to determine their effect on the stability ofbeta-galactosidase (utilizing a ubiquitin-Z-beta-galactosidase chimericfusion) in Saccharomyces cerevisiae, drastic differences were discovered(see Varshavsky (1992) Cell 69: 725-35). For example when Z was met,cys, ala, ser, thr, gly, val, or pro, the resulting polypeptide was verystable (half-life of >20 hours). When Z was tyr, ile, glu, or gln, theresulting polypeptide possessed moderate protein stability (half-life of10-30 minutes). In contrast, the residues arg, lys phe, leu, trp, his,asp, and asn, all conferred low stability on the beta-galactosidasepolypeptide (half-life of <3 minutes). The residue arginine (arg), whenlocated at the amino terminus of a polypeptide, appears to generallyconfer the lowest stability. Thus, chimeric constructs and correspondingfusion polypeptides employing an arg residue at the position Z,described above, are generally preferred embodiments of the presentinvention.

[0247] The above described experiments establishing the relativehalf-lives conferred by each of the 20 possible amino terminal residuesform the basis of the N-end rule. The N-end rule system components arethose gene products which act to bring about the rapid proteolysis ofpolypeptides possessing amino-terminal residues which conferinstability. The N-end rule system for proteolysis in eukaryotes appearsto be a part of the general ubiquitin-dependent proteolytic systempathways possessed by apparently all eucaryotic cells. Briefly, thissystem involves the covalent tagging of a target polypeptide on one ormore lysine residues by a ubiquitin polypeptide marker (to form atarget(lys)-epsilon amino-gly(76) Ubiquitin covalent bond). Additionalubiquitin moieties may be subsequently conjugated to the targetpolypeptide and the resulting “ubiquitinated” target polypeptide is thensubject to complete proteolytic destruction by a large (26S)multiprotein complex known as the proteasome. The enzymes whichconjugate the ubiquitin moieties to the targeted protein include E2 andE3 (or ubiquitin ligase) functions. The E2 and E3 enzymes are thought topossess most of the specificity for ubiquitin dependent proteolyticprocesses.

[0248] A key component of the N-end rule proteolytic pathway in yeast isUBR1 (Bartel, et al. (1990) EMBO J. 9: 3179-89), a gene which encodes anE3 like function which appears to recognize polypeptides possessingsusceptible amino terminal residues and thereby facilitatesubiquitination of such polypeptides (Dohmen et al. (1991) Proc. Natl.Acad. Sci. USA 88: 7351-55). Accordingly UBR1 can be used as aregulatable N-end rule component which is the effector of proteolyticdegradation of the target gene polypeptide. The UBR1 gene has now beencloned from a mammalian organism (Kwon et al. (1998) Proc. Natl. Acad.Sci. USA 95: 7893-903) as well as from yeast. Thus the construction of aUBR1 mouse cell line knockout is imminent and so control of theinstability of Z-Reporter fusions can-be further manipulated bycontrolling the level of UBR1 expressed.

[0249] The UBR1 gene is particularly central to some aspects of thepresent invention because it can be selectively used in conjunction withany of the above described non-methionine “Z” amino-terminaldestabilizing residues including: the most destabilizing—arg; stronglydestabilizing residues—such as lys phe, leu, trp, his, asp, and asn; andmoderately destabilizing residues—such as tyr, ile, glu, or gln. Indeed,it is an object of certain embodiments the present invention to providea means, where desired, to not completely shut-off a negative selectablemarker's function, but merely to attenuate it to some set degree. Thiscan be achieved using the method of the present invention in any of anumber of ways. For example, a moderately destabilizing amino-terminalresidue (Z=tyr, ile, glu, or gln) can be deployed on the targetpolypeptide reporter—resulting in a less rapid removal of the targetpolypeptide pool.

[0250] Other N-end rule components for use in the present inventioninclude S. cerevisiae UBC2 (RAD6), which encodes an E2 ubiquitinconjugating function which cooperates with the UBR1—encoded N-end ruleE3 to promote multiubiquitination and subsequent degradation of N-endrule substrates (Dohmen et al. (1991) Proc. Natl. Acad. Sci. USA 88:7351-55). Thus N-end rule directed proteolysis will not occur in theabsence of either UBR1 or UBC2. This allows either gene to be used asthe inducible “effector of targeted proteolysis” by methods of thepresent invention. Indeed, a target gene polypeptide possessing an N-endrule destabilizing amino-terminal amino acid (such as arg) will bestable until expression of either the UBR1 (E3) or the UBC2 (E2) isinduced from the cognate inducible promoter construct.

[0251] Both UBR1 and UBC2 can be used in conjunction with any of theabove described “Z” amino-terminal destabilizing residues including: themost destabilizing—arg; strongly destabilizing residues—such as lys phe,leu, trp, his, asp, and asn; and moderately destabilizing residues—suchas tyr, ile, glu, or gin. Still other alternative embodiments of theN-end rule component of the present invention are components of theN-end rule system which affect only a subset of the destabilizingresidues. For example, the NTAI deamidase (Baker and Varshavsky (1995) JBiol Chem 270: 12065-74) functions to deaminate amino-terminal asn orgln residues (to form polypeptides with asp or glu amino-terminalresidues respectively). Yeast strains harboring ntal null alleles areunable to degrade N-end rule substrates that bear amino-terminal asn orgln residues. Thus, the NTA1 gene is an alternative embodiment of theN-end rule component of the present invention, but is used preferably inconjunction with a target gene polypeptide (Z-target), in which Z iseither asn or gin. Similarly the ATE1 transferase (Balzi et al. (1990)J. Biol Chem 265: 7464-71) is an enzyme which acts to transfer the argmoiety from a tRNA˜Arg activated tRNA to amino-terminal glu or aspbearing polypeptides. The resulting arg-glu-polypeptide andarg-asp-polypeptide products are then susceptible to the E2/E3—mediatedN-end rule dependent proteolytic processes described above. Thus, theATE1 transferase is an alternative embodiment of the N-end rulecomponent of the present invention, but its use is preferably tied totarget gene polypeptides (Z-target), in which Z is asp, glu, asn or gin.Polypeptides bearing the latter two amino-terminal residues are firstconverted to polypeptides bearing one of the former tow amino-terminalresidues by NTA1 deamidase function described above.

[0252] It is important to note here that, as is the case for therepressor which is made subject to induction by an inducible promoter,the N-end rule component must be available as a clone so that it can beput under the control of an inducible promoter (using standardsubcloning methods known in the art). This can be achieved by firstintroducing genetically engineered copies of the inducible repressor andthe inducible N-end rule component constructs, and subsequently deletingthe normal chromosomal copies of these genes from the host by “knockout”methods. Such methods, we note here are well developed in theart—particularly in the case of both the yeast Saccharomyces cerevisiaeand the mammal mouse. More convenient, however, is the availability of“knock-in” technology which allows the existing chromosomal copy of thegene to be modified to so that its native promoter is deleted and aninducible promoter is inserted in a single step.

[0253] 4.1.3 Ubiquitin Polypeptide Sequences

[0254] A complete and detailed description of the Cub and Nub constructswhich can be used in the method of the present invention is given inU.S. Pat. Nos. 5,503,977 and 5,585,245. A background to the molecularbiology of the ubiquitin proteolytic system in general, and the N-endrule system and ubiquitin sensor association assay is presumed of theskilled artisan seeking to practice the present invention. Briefly,ubiquitin (Ub) is a 76-residue, single-domain protein whose covalentcoupling to other proteins yields branched Ub-protein conjugates andplays a role in a number of cellular processes, primarily through routesthat involve protein degradation. Unlike the branched Ub conjugates,which are formed posttranslationally, linear Ub adducts are thetranslational products of natural or engineered Ub fusions. It has beenshown that, in eukaryotes, newly formed Ub fusions are rapidly cleavedat the Ub-polypeptide junction by Ub-specific proteases (UBPs). In theyeast Saccharomyces cerevisiae, there are at least five species of UBP.Recent work has shown that the cleavage of a Ub fusion by UBPs requiresthe folded conformation of Ub, because little or no cleavage is observedwith fusions whose Ub moiety was conformationally destabilized bysingle-residue replacements or a deletion distant from the site ofcleavage by UBPs.

[0255] The present invention relies in part upon the previouslydescribed split ubiquitin protein sensor system (see U.S. Pat. Nos.5,503,977 & 5,585,245 and WO 02/12902). Briefly, it has beendemonstrated that an N-terminal ubiquitin sub-domain and a C-terminalubiquitin sub-domain, the latter bearing a reporter extension at itsC-terminus, when coexpressed in the same cell by recombinant DNAtechniques as distinct entities, have the ability to associate,reconstituting a ubiquitin molecule which is recognized, and cleaved, byubiquitin-specific processing proteases which are present in alleukaryotic cells. This reconstituted ubiquitin molecule, which isrecognized by ubiquitin-specific proteases, is referred to herein as aquasi-native ubiquitin moiety. As disclosed herein, ubiquitin-specificproteases recognize the folded conformation of ubiquitin. Remarkably,ubiquitin-specific proteases retained their cleavage activity andspecificity of recognition of the ubiquitin moiety that had beenreconstituted from two unlinked ubiquitin sub-domains.

[0256] Ubiquitin is a 76-residue, single-domain protein comprising twosub-domains which are relevant to the present invention—the N-terminalsub-domain and the C-terminal sub-domain. The ubiquitin protein has beenstudied extensively and the DNA sequence encoding ubiquitin has beenpublished (Ozkaynak et al., EMBO J. 6: 1429 (1987)). The N-terminalsub-domain (Nub), as referred to herein, is that portion of the nativeubiquitin molecule which folds into the only alpha-helix of ubiquitininteracting with two beta-strands. Generally speaking, this sub-domaincomprises amino acid residues from about residue number 1 to aboutresidue number 36. The C-terminal sub-domain of ubiquitin (Cub), asreferred to herein, is that portion of the ubiquitin which is not aportion of the N-terminal sub-domain defined in the preceding paragraph.Generally speaking, this sub-domain comprises amino acid residues fromabout 37 to about 76. It should be recognized that by using only routineexperimentation it will be possible to define with precision the minimumrequirements at both ends of the N-terminal sub-domain and theC-terminal sub-domain which are necessary to be useful in connectionwith the present invention.

[0257] It is important to note that the Nub refers, in preferredembodiments of the invention, to the amino-terminal ubiquitin sub-domainunit which has been mutated so as to decrease its binding affinity,thereby making the Cub/Nub association dependent upon the binding of asecond protein pair fused to the Cub and Nub subunits. Suitable forms ofNub are described below and still others are readily available to theskilled artisan by routine mutation and screening methods.

[0258] In order to study the interaction between a hybrid ligand and apair of ligand binding domains, one member of the pair is fused to theN-terminal sub-domain of ubiquitin and the other member of the pair isfused to the C-terminal sub-domain of ubiquitin. Since the members ofthe specific-binding pair (linked to sub-domains of ubiquitin) have anaffinity for the hybrid ligand, this affinity increases the “effective”(local) concentration of the N-terminal and C-terminal sub-domains ofubiquitin, thereby promoting the reconstitution of a quasi-nativeubiquitin moiety. For convenience, the term “quasi-native ubiquitinmoiety” will be used herein to denote a moiety recognizable as asubstrate by ubiquitin-specific proteases. In light of the fact that theN-terminal and C-terminal sub-domains of ubiquitin associate to form aquasi-native ubiquitin moiety even in the absence of fusion of the twosub-domains to individual- members of the ligand binding domain pair, afurther requirement may be imposed in certain embodiments of the presentinvention in order to increase the resolving capacity of the method forstudying such interactions. This further preferred requirement is thatthe N-terminal sub-domain of ubiquitin may be mutation ally altered toreduce its ability to produce, through association with Cub, aquasi-native ubiquitin moiety. It will be recognized by one of skill inthe art that the binding interaction studies described herein arecarried out under conditions appropriate for protein/ligand interaction.Such conditions are provided in vivo (i.e., under physiologicalconditions inside living cells) or in vitro, when parameters such astemperature, pH and salt concentration are controlled in a mannerintended to mimic physiological conditions.

[0259] The mutational alteration of an amino-terminal ubiquitinsub-domain for use with the instant invention is preferably a pointmutation. In light of the fact that it is essential that thereconstituted ubiquitin moiety must “look and feel” like nativeubiquitin to a ubiquitin-specific protease, mutational alterations whichwould be expected to grossly affect the structure of the sub-domainbearing the mutation are to be avoided. A number of ubiquitin-specificproteases have been reported, and the nucleic acid sequences encodingsuch proteases are also known (see e.g., Tobias et al., J. Biol. Chem.266: 12021 (1991); Baker et al., J. Biol. Chem. 267: 23364 (1992)). Itshould be added that all of the at least five ubiquitin-specificproteases in the yeast S. cerevisiae require a folded conformation ofubiquitin for its recognition as a substrate. Extensive deletions withinthe N— sub-domain of ubiquitin are an example of the type of mutationalalteration which would be expected to grossly affect sub-domainstructure and, therefore, are examples of types of mutationalalterations which should be avoided.

[0260] In light of this consideration, the preferred mutationalalteration within the Nub subunit is a mutation in which an amino acidsubstitution is effected. For example, the substitution of an amino acidhaving chemical properties similar to the substituted amino acid (e.g.,a conservative substitution) is preferred. Specifically, the desiredmild perturbation of ubiquitin sub-domain interaction is achieved bysubstituting a chemically similar amino acid residue which differsprimarily in the size of its side chain. Such a steric perturbation isexpected to introduce a desired (mild) conformational destabilization ofa ubiquitin sub-domain. The goal is to reduce the affinity of theN-terminal and C-terminal sub-domains for one another, not necessarilyto eliminate this affinity.

[0261] For example, the mutational alteration may be introduced into theN-terminal sub-domain of ubiquitin. More specifically, a first neutralamino acid residue may be replaced with a second neutral amino acidhaving a side chain which differs in size from the first neutral aminoacid residue side chain to achieve the desired decrease in affinity. Forexample, the first neutral amino acid residue isoleucine (either residue3 or 13 of wild-type ubiquitin) may be replaced with a neutral aminoacids which has a side chain which differs in size from isoleucine suchas glycine, alanine or valine (see Johnsson & Varshavsky, 1994, Proc.Natl. Acad. Sci. U.S.A. 91:10340-10344, the entire contents of which arehereby incorporated by reference).

[0262] A wide variety of fusion construct combinations can be used inthe methods of this invention. One strict requirement which applies toall N- and C-terminal fusion construct combinations is that theC-terminal sub-domain must bear an amino acid (e.g., peptide,polypeptide or protein) extension. This requirement is based on the factthat the detection of interaction between two proteins of interestlinked to two sub-domains of ubiquitin is achieved through cleavageafter the C-terminal residue of the quasi-native ubiquitin moiety, withthe formation of a free reporter protein (or peptide) that hadpreviously been linked to a C-terminal sub-domain of ubiquitin.Ubiquitin-specific proteases cleave a linear ubiquitin fusion betweenthe C-terminal residue of ubiquitin and the N-terminal residue of theubiquitin fusion partner, but they do not cleave an otherwise identicalfusion whose ubiquitin moiety is conformationally perturbed. Inparticular, they do not recognize as a substrate a C-terminal sub-domainof ubiquitin linked to a “downstream” reporter sequence, unless thisC-terminal sub-domain associates with an N-terminal sub-domain ofubiquitin to yield a quasi-native ubiquitin moiety.

[0263] Furthermore, the characteristics of the C-terminal amino acidextension of the C-terminal ubiquitin sub-domain must be such that theproducts of the cleaved fusion protein are distinguishable from theuncleaved fusion protein. In practice, this is generally accomplished bymonitoring a physical property or activity of the C-terminal extensionwhich is cleaved free from the C-terminal ubiquitin moiety. It isgenerally a property of the free C-terminal extension that is monitoredas an indication that a quasi-native ubiquitin has formed, becausemonitoring of the quasi-native ubiquitin moiety directly is difficult ineukaryotic cells due to the presence of native ubiquitin. Whileunnecessary for the practice of the present invention, it would ofcourse be appropriate to monitor directly the presence of thequasi-native ubiquitin as well, provided that this monitoring could becarried out in the absence of interference from native ubiquitin (forexample, in prokaryotic cells, which naturally lack ubiquitin).

[0264] The size of the C-terminal extension which is released followingcleavage of the quasi-native ubiquitin moiety within a reporter fusionby a ubiquitin-specific protease is a particularly convenientcharacteristic in light of the fact that it is relatively easy tomonitor changes in size using, for example, electrophoretic methods. Forinstance, if the C-terminal reporter extension has a molecular weight ofabout 20 kD, the cleavage products will be distinguishable from thenon-cleaved quasi-native ubiquitin moiety by virtue of the appearance ofa previously absent reporter-specific 20 kD band following cleavage ofthe reporter fusion.

[0265] In light of the fact that the cleavage can take place, forexample, in crude cell extracts or in vivo, it is generally not possibleto monitor such changes in molecular weight of cleavage products bysimply staining an electrophoretogram with a dye that stains proteinsnonspecifically, because there are too many proteins in the mixture toanalyze in this manner. One preferred method of analysis isimmunoblotting. This is a conventional analytical method wherein thecleavage products are separated electrophoretically, generally in apolyacrylamide gel matrix, and subsequently transferred to a chargedsolid support (e.g., nitrocellulose or a charged nylon membrane). Anantibody which binds to the reporter of the ubiquitin-specific proteasecleavage products is then employed to detect the transferred cleavageproducts using routine methods for detection of the bound antibody.

[0266] Another useful method is immunoprecipitation of either areporter-containing fusion to C-terminal sub-domains of ubiquitin or thefree reporter (liberated through the cleavage by ubiquitin-specificproteases upon reconstitution of a quasi-native ubiquitin moiety) withan antibody to the reporter. The proteins to be immunoprecipitated arefirst labeled in vivo with a radioactive amino acid such as³⁵S-methionine, using methods routine in the art. A cell extract is thenprepared, and reporter-containing proteins are precipitated from theextract using an anti-reporter antibody. The immunoprecipitated proteinsare fractionated by electrophoresis in a polyacrylamide gel, followed bydetection of radioactive protein species by autoradiography orfluorography.

[0267] A preferred experimental design is to extend the C-terminalsub-domain of ubiquitin with a peptide containing an epitope foreign tothe system in which the assay is being carried out. It is alsopreferable to design the experiment so that the C-terminal reporterextension of the C-terminal sub-domain of ubiquitin is sufficientlylarge, i.e., easily detectable by the electrophoretic system employed.In this preferred embodiment, the C-terminal reporter extension of theC-terminal sub-domain should be viewed as a molecular weight marker. Thecharacteristics of the extension other than its molecular weight andimmunological reactivity are not of particular significance. It will berecognized, therefore, that this C-terminal extension can represent anamalgam comprising virtually any amino acid sequence combination fusedto an epitope for which a specifically binding antibody is available.For example, the C-terminal extension of the C-terminal ubiquitinsub-domain may be a combination of the “ha” epitope fused to mouse DHFR(an antibody to the “HA” epitope is readily available).

[0268] Aside from the molecular weight of the C-terminal amino acidextension of the C-terminal ubiquitin sub-domain, other characteristicscan also be monitored in order to detect cleavage of a quasi-nativeubiquitin moiety. For example, the enzymatic activity of some proteinscan be abolished by extending their N-termini. Such a “reporter” enzyme,which, in its native form, exhibits an enzymatic activity that isabolished when the enzyme is N-terminally extended, can also serve asthe C-terminal reporter linked to the C-terminal ubiquitin sub-domain.

[0269] In this detection scheme, when the reporter is present as afusion to the C-terminal ubiquitin sub-domain, the reporter protein isinactive. However, if the C-terminal ubiquitin sub-domain and theN-terminal ubiquitin sub-domain associate to reconstitute a quasi-nativeubiquitin moiety in the presence of a ubiquitin-specific protease, thereporter protein will be released, with the concomitant restoration ofits enzymatic activity.

[0270] In preferred embodiments, the reporter protein is a eukaryoticnegative selectable marker (NSM) which has been engineered to beprocessed and released as an N-end rule-labile Z-NSM fusion followingubiquitin-specific protease proteolytic cleavage. The negativeselectable markers (NSMs) for use in the invention are describedelsewhere. The advantage of using an Z-NSM fusion is that interaction ofthe specific binding pair can be directly selected for (as opposed toscreened for) by virtue of the fact that only cells in which Z-NSM hasbeen released will survive negative selection.

[0271] The target gene reporter (negative selectable marker) must befused downstream of a codon which encodes an N-end rule susceptibleresidue (Z, as described above) and this residue, in term, must be fusedin-frame to the carboxy-terminus of a ubiquitin coding sequence(generally the carboxy-terminus of a C-terminal ubiquitin sub-domain(Cub) which corresponds to gly76 of intact ubiquitin). The reason forconstructing this extensive chimeric gene construct is to take advantageof the ability of constitutive ubiquitin proteases to cleave any peptidebond which is carboxy-terminal to gly76 of an intact ubiquitin unit.This ubiquitin-specific protease normally functions to processpoly-ubiquitin chains (the translational product of the tandem ubiquitinencoding sequences of eucaryotic genomes) into discrete (normally 76 aa)ubiquitin moieties which are used in ubiquitin-system pathways. In themethod of the present invention, the ubiquitin-specific proteases serveas a convenient means to generate target gene polypeptides bearingspecific amino-terminal residues (Z). Nonetheless, it is understood thatother alternatives to mammalian or yeast ubiquitin exist which canfunction in the method of the present invention. Such ubiquitinequivalents include, for example, ubiquitin mutants, ubiquitin-likeproteins, ubiquitin-related proteins, and ubiquitin-homologous proteins.For example, ubiquitin-like proteins such as NEDD8, UBL1, FUBI, andUCRP, as well as analogous ubiquitin-related proteins such asSUMO/Sentrin/Pic1 may be used as ubiquitin equivalents in the method ofthe invention. Other proteins related to ubiqutin, but which aresomewhat less homologous to it, include ubiquitin-homologous proteinssuch as Rad23 and Dsk2 whose similarity to ubiquitin does not includethe presence of a carboxyl-terminal pair of glycines. Theseubiquitin-like proteins share the common features of being related toubiquitin by amino acid sequence homology and, with the apparentexception of the ubiquitin homologous proteins, of being covalentlytransferred to cellular protein targets post-translationally.

[0272] Indeed, in some embodiments the intended scope of the immediateinvention encompasses any means known in the art by which a targetpolypeptide bearing an N-end rule susceptible residue (Z=arg, lys, his,leu, phe, try, ile, trp, asn, gln, asp, or glu) can be generated.General methods for engineering such N-end residues intoubiquitin-reporter chimera expression vectors are well known in the art(e.g. the “fusion PCR” method; see Karreman (1988) BioTechniques 24:736-42).

[0273] The summary description in the preceding paragraph does notdiscuss certain important experimental considerations. For example, fortwo interacting proteins, P1 (fused to Nub) and P2 (fused to Cub) thefollowing additional considerations are included within the scope of theinvention. In light of its role as an affinity component, it will berecognized that P1 can be fused to the N-terminus or the C-terminus ofthe N-terminal ubiquitin sub-domain. Similarly, P2 can be fused to theN-terminus or the C-terminus of the C-terminal ubiquitin sub-domain. IfP2 is fused to the C-terminus of the C-terminal ubiquitin sub-domain, itwill be removed by cleavage by the ubiquitin-specific protease,providing that the ubiquitin sub-domains associate to form aquasi-native ubiquitin moiety. Consistent with the summary descriptionin the preceding paragraph, if the P2 moiety is fused to the C-terminusof the C-terminal ubiquitin sub-domain, it may also be used as areporter for detecting reconstitution of a quasi-native ubiquitinmoiety. Furthermore, the position of P2 within the C-terminalreporter-containing region of the fusion is not a criticalconsideration.

[0274] 4.1.4 Detection of Cleavage of the Reporter Moiety

[0275] The most straight forward way to detect cleavage of the reportermoiety is by detecting the presence of the cleaved “free-RM”. Oneroutine assay for that type of detection is achieved by Western blotusing an antibody specific for the RM. No additional activity of the RMis required as long as it is reasonably stable. For that reason, a Metshall be present at the N-terminus of the cleaved RM. Alternatively, ifthe N-terminus of the cleaved RM has a non-stabilizing amino acid andthe free-RM form will therefore be degraded, a detection of theun-cleaved RM linked to Cub will also be able to assess the degree ofcleavage which has occurred. To obviate the need of an antibody for eachparticular RM, an epitope tag (such as HA, myc, or any other routinelyused tags against which commercially available antibodies may exist) maybe fused to the RM at a proper location, such as the C-terminus. Westernblot is well-known in the art and can be found in a number of laboratorymanuals.

[0276] If the RM has an enzymatic activity that is only present when theRM is cleaved off the Cub-RM fusion, degree of cleavage can also beindirectly determined by assaying for the enzymatic activity of the freeRM. For example, some kinases my be inactive when fused to an N-terminalinhibitory domain and become activated after removing the inhibitorydomain. Such kinases can be used as a RM for this embodiment of theinvention. A Met shall preferably form the N-terminus of the free-RM.

[0277] Similarly, if a RM is enzymatically inactivated/degraded when itis cleaved off the fusion, an assay of the enzymatic activity can alsobe used to determine the degree of cleavage. For that assay, a non-Metamino acid is preferably the first amino acid of the cleaved RM.

[0278] Other activities of the RM may be useful for detecting cleavage.For example, if the RM is a fluorescent protein, then the cleaved RM maybe degraded by UBP if the first amino acid is non-Met. Changes influorescent strength can be measured to indicate the degree of cleavage.

[0279] If the RM is a transcription factor (e.g. PLV, Stagljar et al.(1998) Proc. Natl. Acad. Sci. U.S.A., 95: 5187-92), cleaved RM may nowrelocate to the nucleus and be available for transcriptional activationof a reporter gene, the activity of which in turn serves as an indicatorof the degree of cleavage. If the un-cleaved RM is able to serve as atranscription factor, then the overall level of transcription isexpected to drop if the cleaved free-RM is unstable as determined byN-end rule.

[0280] The above exemplary detection methods are for illustrationpurpose only. A skilled artisan shall be able to envision equivalentmethods of these examples, and thus, those equivalent methods are alsowithin the scope of the instant invention.

[0281] 4.2 Other Reporter Systems

[0282] According to the invention, a transcription based reporter systemcan be used to detect whether P1 and P2 are within close range of eachother. A typical transcription-based reporter system is yeast two-hybridsystem, which is well-known in the art (see below). In that respect, P1and P2 are both synthesized as fusion proteins, one fused to a DNAbinding domain, the other fused to a transcription activation domain.The DNA binding domain will bind to the promoter region of a reportergene. If P1 and P2 are with close range of each other (via binding toR1-Y-R2), then the transcription activation domain will be able toactivate the transcription of a reporter gene, which will facilitate theidentification of either the test protein or the test small chemicalcompound. Due to the symmetric nature of the system, there shall be nolimitation as to whether P1 or P2 is fused to the DNA binding domain orthe transcription activation domain. In addition, both P1 and P2 can besynthesized as either N- or C-terminal fusion proteins.

[0283] Detailed description of various components of yeast two hybridsystem can be readily found elsewhere. For example, The Yeast Two-HybridSystem (Advances in Molecular Biology), Ed. Paul L. Bartel and StanleyFields, Oxford University Press, 1997, is a book devoted solely to theyeast two-hybrid system. Pioneers in the field provide detailedprotocols, practical advice on troubleshooting, and suggestions forfuture development. In addition, they illustrate how to construct anactivation domain hybrid library, how to identify mutations that disruptan interaction, and how to use the system in mammalian cells. Chaptertopics include characterizing hormone/receptor complexes; identifyingpeptide ligands; and analyzing interactions mediated by proteinmodifications. Equally valuable two-hybrid techniques and variations canalso be found in Yeast hybrid technologies (Zhu, L., and Hannon, G. J.,Eds., Biotechniques Press, Westborough, Mass., USA, 2000). A third book,Two-Hybrid Systems: Methods and Protocols (Methods in Molecular BiologyVol. 177), Ed. Paul MacDonald, Humana Press, 2001, provides some recentupdates to the field of yeast two-hybrid assay.

[0284] Other version of yeast two-hybrid systems are also described. Forexample, the reverse yeast two-hybrid system is described in U.S. Pat.Nos. 5,955,280 and 5,965,368, the contents of which are incorporatedherein in their entirety. These patents disclosed methods foridentifying molecular interactions (e.g., protein/protein, protein/DNA,protein/RNA, or RNA/RNA interactions), all of which employ selection andcounter-selection and at least two hybrid molecules. Similar to theconventional yeast two-hybrid system, reverse two-hybrid systems alsoinvolve molecules which interact to reconstitute a transcription factorand direct expression of a reporter gene, the expression of which isthen assayed. Also disclosed by these patents are genetic constructswhich are useful in practicing the methods of the invention.

[0285] Licitra and Liu (WO 97/41255, and U.S. Pat. No. 5,928,868) alsodescribed a “three hybrid screen assay” in which the basic yeasttwo-hybrid assay system is implemented. The significant difference is:instead of depending on the interaction between a so-called “bait” and aso-called “prey” protein, the transcription of the reporter gene isconditioned on the proximity of the two proteins, each of which can bindspecifically to one of the two moieties of a small hybrid ligand. Thesmall hybrid ligand constitute the “third” component of the hybrid assaysystem. In that system, one known moiety of the hybrid ligand will bindto the “bait” protein, while the interaction between the other moietyand the “prey” protein can be exploited to screen for either a proteinthat can bind a known moiety, or a small moiety (pharmaceutical compoundor drug) that can bind a known protein target.

[0286] Bartel and Fields summarize many different approaches/variationsof the available two-hybrid systems in The yeast-two-hybrid system(Bartel, P. L., and Fields, S., Eds., Oxford University Press, New York,N.Y., USA, 1997). Equally valuable two-hybrid techniques and variationscan also be found in Yeast hybrid technologies (Zhu, L., and Hannon, G.J., Eds., Biotechniques Press, Westborough, Mass., USA, 2000). Furthersystems include WO 9602561, a two hybrid system using conformationallyconstrained proteins as one of the hybrids; EP 0646644, a periplasmicmembrane bound interaction system; WO 98/25947, a prokaryotic two-hybridsystem using E. coli and other cells; and WO 98/07845, an interactiontrap system or “ITS” which is derived using recombinantly engineeredprokaryotic cells. WO 98/34120 describes a strategy for designing andimplementing protein-fragment complementation assays (PCAs) to detectbiomolecular interactions in vivo and in vitro—the DHFR proteininteraction screening system. The design, implementation and broadapplications of this strategy are illustrated with a large number ofenzymes with particular detail provided for the example of murinedihydrofolate reductase (DHFR). Fusion peptides consisting of N andC-terminal fragments of murine DHFR fused to GCN4 leucine zippersequences were coexpressed in Escherichia coli grown in minimal medium,where the endogenous DHFR activity was inhibited with trimethoprim.Coexpression of the complementary fusion products restored colonyformation. Survival only occurred when both DHFR fragments were presentand contained leucine-zipper forming sequences, demonstrating thatreconstitution of enzyme activity requires assistance of leucine zipperformation. DHFR fragment-interface point mutants of increasing severity(Ile to Val, Ala and Gly) resulted in a sequential increase in E. colidoubling times illustrating the successful DHFR fragment reassemblyrather that non-specific interactions between fragments. This assaycould be used to study equilibrium and kinetic aspects of molecularinteractions including protein-protein, protein-DNA, protein-RNA,protein-carbohydrate and protein-small molecule interactions, forscreening cDNA libraries for binding of a target protein with unknownproteins or libraries of small organic molecules for biologicalactivity. The selection and design criteria applied here is developedfor numerous examples of clonal selection, colorometric, fluorometricand other assays based on enzymes whose products can be measured. Thedevelopment of such assay systems is shown to be simple, and providesfor a diverse set of protein fragment complementation applications. WO98/39483 shows methods for identifying nucleic acid sequences thataffect a cellular phenotype. The method uses a reporter gene whose levelof expression correlates with the phenotype in conjunction with a methodor device for measuring the level of reporter expression. WO 98/44350discloses an enzyme complementation assay in which methods andcompositions for detecting molecular interactions, particularlyprotein-protein interactions, are provided. The invention allowsdetection of such interactions in living cells or in vitro. Detection ofmolecular interactions in living cells is not limited to the nuclearcompartment, but can be accomplished in the cytoplasm, cell surface,organelles, or between these entities. In one embodiment, the methodutilizes novel compositions comprising fusion proteins between themolecules of interest and two or more inactive, weakly-complementingβ-galactosidase mutants. Association between the molecules of interestbrings the complementing β-galactosidase mutants into proximity so thatcomplementation occurs and active β-galactosidase is produced. Theactive β-galactosidase may be detected by methods well-known in the art.A further similar assay was disclosed in WO 99/28746 and relatedapplication WO01/73108; this assay was designed to perform specificallyin bacterial expression systems, and it relies on the activation, ratherthan functional complementation, of an enzymatic activity by the spatialproximity of two fragments induced by an interaction of a molecule ofinterest with a test substance each fused or bound to one of thefragments. Van Ostade et al., J. Interf. Cytok. Res. (2000), 20, 79-87,WO0/06722 and WO 01/90188 collectively suggest a bioassay for ligandsthat signal through receptor clustering, called MAPPIT. Specifically,the invention relates to a recombinant receptor, comprising anextracellular ligand-binding domain and a cytoplasmic domain thatcomprises a heterologous bait polypeptide, which receptor is activatedby binding of a ligand to said ligand binding domain and by binding of aprey polypeptide to said heterologous bait peptide. The invention alsorelates to a method to detect compound-compound binding using saidrecombinant receptor. WO 94/18317, WO 96/13613, WO 99/41258 (Schreiber,methods to induce a biological event by compound induced dimerization),and Ghosh et al., J. Am. Chem. Soc., 2000, 122: 5658-9 (reconstitutionof fluorescence from a split green fluorescent protein).

[0287] Systems for studying protein-protein interactions in mammaliancells have also be described. For example, Fearon et al. (Karyoplasmicinteraction selection strategy: A general strategy to detectprotein-protein interactions in mammalian cells, Proc. Natl. Acad. Sci.USA 89: 7958-7962, 1992) describe a strategy and reagents for study ofprotein-protein interactions in mammalian cells, termed the karyoplasmicinteraction selection strategy (KISS). With this strategy, specificprotein-protein interactions are identified by reconstitution of thefunctional activity of the yeast transcriptional activator GAL4 and theresultant transcription of a GAL4-regulated reporter gene.Reconstitution of GAL4 function results from specific interactionbetween two fusion proteins: one contains the DNA-binding domain ofGAL4; the other contains a transcriptional activation domain.Transcription of the reporter gene occurs if the two fusion proteins canform a complex that reconstitutes the DNA-binding and transcriptionalactivation functions of GAL4. Using the KISS system, Fearon et al.demonstrate specific interactions for sequences from three differentpairs of proteins that complex in the cytoplasm. In addition, theydemonstrate that reporter genes encoding cell surface or drug-resistancemarkers can be specifically activated as a result of protein-proteininteractions. With these selectable markers, the KISS system can be usedto screen specialized cDNA libraries to identify novel proteininteractions.

[0288] In an extension of the work of Michnick et al. (WO 98/34120) andRossi et al. (WO 98/44350), Wehrman et al., Proc. Natl. Acad. Sci.U.S.A. (2002), 99:3469-3474 recently described an enzyme complementationassay employing a-197 and ω-198 fragments of a class A β-lactamase. Theβ-lactamases are small, monomeric enzymes that can be expressed both inprokaryotes and eukaryotes, including mammalian cells. Particularly inthe latter, where no constitutive expression of β-lactamases is present,this resulted in a highly sensitive system with low background. Asimilar system was also recently described in WO 01/94617 and inGalarneau et al., Nat. Biotech. (2002), 20:619-622. Surprisingly, thesesystems are equally well suited to be used in a three hybrid mode usingthe hybrid ligands and methods of the present invention. Thereby, amethod to determine whether a polypeptide P2 and a small molecule R2 areable to bind each other is created.

[0289] Such an embodiment will preferably comprise the use of eukaryoticcells, more preferably mammalian cells, although in certain casesbacterial cells, fungal cells, plant cells or insect cells may bepreferred and are not excluded. In order to obtain good signal-to-noiseratios, the host cell type of choice should not contain constitutivelyexpressed β-lactamases. The β-lactamase fragments may be taken from aclass A β-lactamase, but may also be taken from another class, and thefragments may comprise amino acid substitutions, additions or deletionscompared to the wild type protein. For example, Wehrman et al., Proc.Natl. Acad. Sci. U.S.A. (2002), 99:3469-3474 have shown that theaddition of a Asn-Gly-Arg tripeptide to the carboxy terminal end of theα-197 fragment produced a profound enhancement of the activity of thereconstituted enzyme.

[0290] For this assay, the protein P2 for which a small moleculeinteractor is sought, or a library of proteins where an interactingpartner is sought for a small molecule ligand R2, is cloned into anexpression vector in frame with one of the β-lactamase fragments, thepolypeptide known to interact with R1, e.g. DHFR where R1 ismethotrexate, is cloned into an expression vector in frame with thesecond of the β-lactamase fragments. Any type of expression vector mayin principle be used, such as, for example, a plasmid, cosmid, phagevector or artificial chromosome, and the choice will depend on the hostcell type to be employed. For example, for expression in mammaliancells, a retroviral vector may be applied generating stably transfectedcells; in other instances, it may be advantageous to transfect onlytransiently. The vectors preferably further carry a selectable marker,for example an antibiotic resistance or auxotrophic marker, which arepreferably different for the two vectors encoding the two differentβ-lactamase fragments. After construction, the vectors are introducedinto the host cells by a method to be chosen depending on the host celltype, such as electroporation, lipofection, rendering cells chemicallycompetent by heat shock or treatment with bivalent cations, packaging ofDNA or RNA into viral or phage particles and subsequent transduction ofhost cells or with ballistic methods (“gene gun”).

[0291] After introduction of the vectors encoding the β-lactamasefragments into host cells, the host cells are preferably grown underconditions conducive to growth only for cells containing both selectablemarkers encoded by the two β-lactamase fragment encoding vectors. Forexample, without limitation, if one of the vectors encodes a hygromycinresistance, the other a HIS3 marker, then the host cells will becultured in a medium containing hygromycin but lacking histidine.

[0292] Ultimately, the host cells, or their progeny, are treated withthe hybrid ligand, or hybrid ligands, and the activity of theβ-lactamase is assayed in the cells in the presence of the hybridligand(s). This step may be carried out on whole cells, but it may alsoprove advantageous to permeabilize or lyse the cells before addition ofthe hybrid ligand(s) and/or the β-lactamase substrate. The choice of theβ-lactamase substrate will in part depend on the host cell system, andthe desired method of detection. Numerous suitable substrates are knownto the skilled person, such as nitrocefin (detection of absorbance ofvisible light) or CCF2/AM (detection of fluorescence). The β-lactamantibiotics could be used for a growth inhibition assay when host cellsare bacteria.

[0293] A skilled artisan shall be able to identify the suitabletwo-hybrid system components for use with the instant invention withoutundue experimentation. These will include, but are not limited toexpression vectors for reporter systems and their assay/detectionmethods, expression vectors for expression of fusion proteins comprisingthe two moieties collectively constituting the reporter system andP1/P2, respectively. In certain embodiments, P2 is from a polypeptidelibrary or libraries, so the vector chosen for the expression of the P2fusion shall be appropriate for library construction. A skilled artisanshall be able to utilize any of the technologies/methods describedabove, or combination thereof, or modification thereof, to practice theinstant invention. The contents of all these references are incorporatedby reference herein.

[0294] 4.3 Reporter Genes

[0295] In a reporter system based on the transcriptional activation of areporter gene, one has to choose a reporter gene appropriate for thehost cell type and assay format envisaged. The host cell of choice needsto provide the appropriate transcriptional machinery, the choice ofreporter gene will depend on the method chosen to detect and potentiallyquantify the transcription of the reporter gene, for example, by WesternBlot, calorimetric or fluorimetric methods or a growth inhibition assayon selective or counterselective media, or a cell surface marker.

[0296] A wide range of reporter genes suitable for use in the methods ofthe present invention will be known to the skilled artisan, and he willbe readily able to chose the appropriate reporter gene for a given assayformat. Such reporter gene may be a positive selectable marker genewhich can be selected for under appropriate conditions. In principle,any non-redundant gene in a synthetic pathway that is essential to thesurvival of the cell can be used for the construction of an auxotrophicpositive selectable marker, but frequently used such makers include,without limitation, HIS3, LYS2, LEU2, TRP2, ADE2. Usually, a cell lineis constructed that is deficient in the marker gene, and that can onlygrow on media supplemented with the corresponding metabolic product,i.e. histidine, lysine, leucine, tryptophane or adenine. When used forselection, a desirable phenotype, i.e. expression of a desiredrecombinant gene, is linked to the expression of the gene the cell isdeficient in. Other positive selectable markers include antibioticresistance markers, e.g. Hygromycin resistance (HygR), neomycinresistance (neoR), puromycin resistance (PACR) or Blasticidin Sresistance (BlaSR), or any other antibiotic resistance marker. Here,expression of a desired recombinant gene is linked to the expression ofthe antibiotic resistance marker by transforming cells with geneconstructs comprising both the desired recombinant gene and arecombinant form of the antibiotic resistance marker gene. Selection isthen carried out on media containing the antibiotic, e.g. Hygromycin,neomycin, puromycin or Blasticidin S.

[0297] In addition, the reporter gene may encode a detectable proteinthat, upon transcriptional activation of said reporter gene, allows hostcells to be visually differentiated from host cells in which saidreporter gene has not been activated. Such a detectable protein ispreferably encoded by at least one of the genes lacZ, gfp, yfp, bfp,cat, luxAB, HPRT or a cell surface marker gene. Other similar genesexist and the person skilled in the art will readily identify other suchgenes that can be employed according to this embodiment.

[0298] WO 98/25947 describes a prokaryotic two-hybrid assay system,which also provides details about bacterial reporter genes that can beused with the instant invention. The contents of WO 98/25947 areincorporated by reference herein. Selectable markers for use inbacterial cells include antibiotic resistance markers, e.g. bla(beta-lactamase resistance gene), cam (chloramphenicol acetyltransferase gene) or kan (kanamycin phosphoryl transferase gene),luminescence markers such as gfp, color inducing markers, for examplelacZ, auxotrophic markers (any amino acid biosynthesis gene) and heavymetal resistance markers. Further selectable markers may be found in:Escherichia coli and Salmonella: Cellular and molecular biology, Secondedition, F. C. Neidhardt, et al. (Edrs.), 1996. ASM Press, Washington,D.C., USA

[0299] Furthermore, negative selectable reporter genes which can be usedin a cell, and which can be selected against under appropriateconditions, may be employed. In preferred applications, the reporter isa selectable marker which is capable of both positive and negativeselection. For example, the reporter gene may be chosen from the list ofURA3, HIS3, LYS2, HygTk, Tkneo, TKBSD, PACTK, HygCoda, Codaneo, CodaBSD,PACCoda, Tk, codA, and GPT2. The reporter moiety may also be TRP1, CYH2,CANI, HPRT.

[0300] A preferred example of a negative selectable marker gene for usein yeast is the URA3 gene which can be both selected for (positiveselection) by growing ura3 auxotrophic yeast strains in the absence ofuracil, and selected against (negatively selection) by growing cells onmedia containing 5-fluoroorotic acid (5-FOA) (Boeke, et al., 1987,Methods Enzymol 154: 164-75). The concentration of 5-FOA can beoptimized by titration so as to maximally select for cells in which theURA3 reporter is inactivated by proteolytic degradation to somepreferred extent. For example, relatively high concentrations of 5-FOAcan be used which allow only cells expressing very low steady-statelevels of URA3 reporter to survive. In contrast, lower concentrations of5-FOA can be used to select for binding partners with relatively weakaffinities for one another. In addition, proline can be used in themedia as a nitrogen source to make the cells hypersensitive to the toxicaffects of the 5-FOA (McCusker & Davis (1991) Yeast 7: 607-8).Accordingly, proline concentrations, as well as 5-FOA concentrations canbe titrated so as to obtain an optimal selection for URA3 reporterdeficient cells. Therefore the use of URA3 as a negative selectablemarker allows a broad range of selective stringencies which can beadapted to minimize false positive background noise and/or to optimizeselection for high affinity binding interactions. Other negativeselectable markers which can be adapted to the methods of the inventionare included within the scope of the invention.

[0301] Another example of a negative selectable marker gene for use inyeast is the TRP1 gene which can be both selected for (positiveselection) by growing trp1 auxotrophic yeast strains in the absence oftryptophan, and selected against (negatively selection) by growing cellson media containing 5-fluoroanthranilic acid (5-FAA) (Toyn et al., 2000,Yeast, 16: 553-560).

[0302] Two other negative selectable marker genes for the use in yeastare CYH2 and CANI both of which can be selected against (negativeselection) by growing cells on media containing cycloheximide orcanavanine (The Yeast Two-Hybrid System (Advances in Molecular Biology),Ed. Paul L. Bartel and Stanley Fields, Oxford University Press, 1997).

[0303] Counter-selectable markers for use in bacteria include sacB (B.subtilis gene encoding levansucrase that converts sucrose to levans,which is harmful to the bacteria), rpsL (strA) (Encodes the ribosomalsubunit protein (S12) target of streptomycin), tetA^(R) (Confersresistance to tetracycline but sensitivity to lipophilic compounds, e.g.fusaric and quinalic acids), phe^(S) (Encodes the subunits of Phe-tRNAsynthetase, which renders bacteria sensitive to p-chlorophenylalanine, aphenylalanine analog), thyA Encodes thymidilate synthetase, whichconfers sensitivity to trimethoprim and related compounds, lacY (Encodeslactose permease, which renders bacteria sensitive tot-o-nitrophenyl—D-galactopyranoside), gata-1 (Encodes a zinc fingerDNA-binding protein which inhibits the initiation of bacterialreplication), ccdB (Encodes a cell-killing protein which is a potentpoison of bacterial gyrase). Further counter-selectable markers may befound in: Escherichia coli and Salmonella: Cellular and molecularbiology, Second edition, F. C. Neidhardt, et al. (Edrs.), 1996. ASMPress, Washington, D.C., USA

[0304] Numerous selectable markers which operate in mammalian cells areknown in the art and can be adapted to the method of the invention so asto allow direct negative selection of interacting proteins in mammaliancells. Examples of mammalian negative selectable markers includeThymidine kinase (Tk) (Wigler et al., 1977, Cell 11: 223-32; Borrelli etal., 1988, Proc. Natl. Acad. Sci. USA 85: 7572-76) of the Herpes Simplexvirus, the human gene for hypoxanthine phosphoriboxyl transferase (HPRT)(Lester et al., 1980, Somatic Cell Genet. 6: 241-59; Albertini et al.,1985, Nature 316: 369-71) and Cytidine deaminase (codA) from E. coli(Mullen et al., 1992, Proc. Natl. Acad. Sci. USA 89: 33-37; Wei andHuber, 1996, J. Biol. Chem. 271: 3812-16). For example: the Tk gene canbe selected against using Gancyclovir (GANC) (e.g. using a 11Mconcentration) and codA gene can be selected against using 5-FluorCytidin (5-FIC) (e.g. using a 0.1-1.0 mg/ml concentration). In addition,certain chimeric selectable markers have been reported (Karreman, 1998,Gene 218: 57-61) in which a functional mammalian negative selectablemarker is fused to a functional mammalian positive selectable markersuch as Hygromycin resistance (Hyg^(R), neomycin resistance (neo^(R)),puromycin resistance (PAC^(R)) or Blasticidin S resistance (BlaS^(R)).These produce various Tk-based positive/negative selectable markers formammalian cells such as HygTk, Tkneo, TKBSD, and PACTk, as well asvarious codA-based positive/negative selectable markers for mammaliancells such as HygCoda, Codaneo, CodaBSD, and PACCoda. Tk-neo reporterswhich incorporate luciferase, green fluorescent protein and/orbeta-galactosidase have also been recently reported (Strathdee et al.,2000, BioTechniques 28: 210-14). These vectors have the advantage ofallowing ready screening of the “positive” marker/reporter byfluorescent and/or immunofluorescent microscopy. The use of suchpositive/negative selectable markers affords the advantages mentionedabove for URA3 as a reporter in yeast, inasmuch as they allow mammaliancells to be assessed by both positive and negative selection methods forthe expression and relative steady-state level of the reporter fusion.For example, Rojo-Niersbach et al reported the use of GPT2 (GuaninePhosphoryl Transferase 2) in mammalian cells as a basis for theselection of protein interactions (Biochem. J. 348: 585-590, 2000).

[0305] The above listing of genes suitable for use as reporter genes inthe methods of the present invention is not meant to be exhaustive norlimiting. The skilled artisan may know other or become aware of newlydiscovered or developed systems suitable for use as reporter genes inthe methods of the present invention. The scope of the present inventionis meant to include their use.

[0306] 4.4 The Halo Growth Assay

[0307] A halo growth assay may be used in several embodiments of thepresent invention. Generally, this type of assay provides for thequalitative determination of the effect of different concentrations of acompound on cellular growth. In essence, a halo growth assay comprisesthe distribution of a dilute solution of the cells under investigationon an agar plate, followed by the placement of a drop of a solutioncontaining the compound under investigation on a predetermined spot onthe agar (for example the middle of a petri dish). Subsequently, theagar plate is cultured under conditions conducive to cellular growth,and growth is assessed a predetermined time later. During this time, thecompound will diffuse through the agar, forming a concentration gradientwith its highest concentration at the point of application, radiallydeclining outwards from this point. If the agar is prepared to sustaincellular growth, and the compound has no effect, a uniform cell carpetshould be found. Conversely, if the agar is prepared to stifle cellulargrowth, for example agar lacking a component essential for cellulargrowth, and the compound has no effect, no cell growth should appear. Ifthe compound has a toxic effect on the cells, no change should be seenwith growth-stifling agar, but on growth-sustaining agar, a circulararea (Halo) without growth should appear on growth-sustaining agararound the point of application, growth gradually declining inwards tothis point. Where a compound has a beneficial effect on growth, such ascomplementing the lack of an essential component in a growth stiflingagar, a circular Halo of growth should appear around the point ofapplication, growth gradually declining outwards from this point. Suchhalo assays will be familiar to a skilled artisan. However, alternativemethods fulfilling the same needs may be used equivalently.

[0308] In certain embodiments of the invention, it may be advantageousto conduct large numbers of such assays for a single experiment,preferably greater than about 10, 100, 1 000 or more than 10 000 assays.Such numbers of assays may be assisted through the use of petri or agardishes of around 70, 300, 480 or greater than 500 cm² surface area on towhich the cells and hybrid ligand/compounds of the invention are placed.Indeed, to maximise throughput and minimise the cost of performing asingle such assay, it is preferable to reduce the scale of the assay.Minimised assays may for example, be conducted using microtitre plate ofpreferably 96, 384, 1536 or more than 1536 wells. Alternatively, suchassays may be conducted on solid growth agar where the cells and hybridligand/compounds are placed at high numbers or densities. For example,around 10, 100, 1 000 or more than 10 000 separate assays may beconducted on one or more petri or agar dishes, wherein one particularassay is separated from another assay by a distance of about 1, 3, 10 ormore than 30 mm. In certain embodiments, it is advantageous that theassays are placed in a regular pattern so that subsequent analysis ofgrowth can be more readily conducted by eye or machine vision. Suchnumbers, densities or patterns of assays may be formed by a number ofmethods, as will be apparent to a person skilled in the art. Forexample, 8, 12 or 16-way multi channel pipettes or 96384-wellreplicators (Genetix) may be used. Alternatively, if high throughout oraccuracy is desired, an automated device may be employed. Many suitableautomated devices will be known to the skilled artisan and included without limitation automated pipetting units with 1, 2, 4, 8, 12, 96 or morethan 96 pipetteing tips such as sold by several manufacturers includingthe MultiProbe II or MultiTrack (Packard), Hamillton, Quadra 96 or 384(Tomtec), CyBio etc. Other automated devices that accurately transferlarge numbers of small amounts of biologically active materials my alsobe employed. For example, gridding robots such as the Qbot (Genetix,UK), BioGrid (BioRobotics, UK) or those described in Maier et al 1997(in Automation for genome characterisation. Ed T J Beuelsdijk. J WileyNew York) may be employed.

[0309] 4.5 The Fluorescence Detection Growth Assay

[0310] A growth assay which can be performed in a microtiter plateformat is advantageous. For example, MTPs can be easily handled in largenumbers, use relatively little material per assay and hence largenumbers of assays may be conducted using standard laboratory automation.We developed such an assay based on the principle that cells growing insuspension consume oxygen from the surrounding medium. However, usingthis principle is not meant as limiting the scope of the invention, asthe skilled person will be able to appreciate other methods of assessingthe growth of cells in microtiter plates.

[0311] With an integrated oxygen sensor built into the bottom of theplate, the OxoPlate (PreSens Precision Sensing GmbH, Regensburg,Germany) is able to measure the oxygen concentration in the solution ineach well of a 96 well plate in near-real time (response time <30 s).The measurement is based on the fluorescence emission of two dyes in asensor on the bottom of each well, one of which can be quenched be byoxygen, while the fluorescence of the second dye is unaffected byoxygen, and is used as an internal reference. Both dyes have equalexcitation (540 nm), but different Stokes shifts and emissionwavelengths (quenchable dye: 590 nm, unquenchable dye: 650 nm). Theratio of the emissions at 650 nm and 590 nm(I_(quenchable)/I_(unquenchable)) is taken as a measure of oxygenconcentration. When the oxygen partial pressure in the solution in thewell is reduced, the emission intensity of the dye that can be quenchedby oxygen will rise, while the emission intensity of the second dye willremain constant. Using such internal reference makes this assayindependent of many potential error sources, such as instability of theoptical system. It also obviates the need for separate calibrationwells, and hence all 96 wells of a 96 well plate can be used forsamples. This method uses a plate reader which can read from the bottomof a microtiter plate, and can measure in dual kinetic mode, i.e. takingseveral measurement at two different wavelengths. Suitable readers willbe well known to a person skilled in the art and include withoutlimitation the Perkin Elmer Wallac Victor2 V 1420 multilabel HTS counter(Perkin Elmer, Wellesley, Mass., USA).

[0312] When suitable cells are seeded into the wells of an OxoPlate in amedium conducive to growth, logarithmic cell growth will occur, oxygenwill be used up and the oxygen partial pressure may become limiting. Asthe level of oxygen diminishes further, cell growth could becomehampered, until the oxygen partial pressure reaches near-zero at whichpoint cell growth may cease. This growth pattern is reflected in asigmoidal curve of the fluorescence emission intensity ratio of the twodyes. Conversely, if the medium in a well stifles growth, no oxygen willbe used, and the measurements of the fluorescence emission intensityratio yield a constant line near the value for medium without cells.

[0313] 5. Hybrid Small Molecules

[0314] Yeast three hybrid assays using hybrid ligand compounds differentfrom those of the present invention are known in the art (See, forexample: Crabtree et al. WO 9418317; Schreiber et al. WO 9613613; Holtet al. WO 9606097; Licitra and Liu WO 9741255; Bergmann et al., J.Steroid Biochem. Molec. Biol. 1994, 49:139-52; Lin et al., J. Am. Chem.Soc. 2000, 122:4247-8). However, the hybrid ligand compounds accordingto the present invention possess advantageous properties setting themdistinctly apart from those described in the prior art. For example, Linet al. used a metadibenzothioester as linker between R1 and R2,conferring rigidity, lipophilicity and low water solubility to theirMtx-mdbt-Dex hybrid ligand compound. In order to pass cell membranes, acertain lipophilicity is desirable. However, in order to get to themembrane, such compound first has to cross an aequeous compartment bydiffusion. If its water solubility is too low, too little compound canreach the membrane and exert its effect inside the cell.

[0315] 5.1 Linker Sequences

[0316] In certain embodiments, any chemical linker Y (includingsynthetic polypeptides, see below) can be used to link R1 to R2,provided that the presence of the linker sequence will not significantlyinterfere with the reporter system when P1 binds to R1 and P2 binds toR2. In addition, the presence of the linker should not overly adverselyaffect the affinities between P1 and R1 or between P2 and R2.

[0317] As such, in order to confirm the suitability of a given hybridligand as a dimerizing compound of general structure R1-Y-R2 for theuses proposed herein, it may be helpful to characterize the bindingproperties of such hybrid ligand to its binding partners P1 and P2, inas far as these are known, and to possibly compare these bindingcharacteristics with those of the unlinked compounds R1 and R2,respectively. Preferably, the hybrid ligand should exhibit bindingproperties similar to the binding properties of the unlinked compounds.However, the molecular weight increase brought about by the linking, aswell as steric and electronic effects caused by the attachment of thelinker to a functional group of the unlinked compounds may alter thebinding characteristics. Therefore, while not being essential, it ispreferable to perform such characterization on a newly synthesizedhybrid ligand. This, however, should not be interpreted as limiting thescope of the invention.

[0318] The affinity of hybrid ligands to their corresponding bindingpartners may be determined, for example, using a BIACORE™ assay system(Biacore AB, Uppsala, SE). Other systems yielding a qualitativelysimilar result, for example, those developed by Affinity Sensors(Cambridge, UK), will be readily apparent to those skilled in the art.Furthermore, other interaction methodologies that measure the bindingaffinities between a hybrid ligand and its binding proteins may beemployed.

[0319] Linker moieties (Y), need not contain essential elements forbinding to the PI and/or P2 proteins, and for certain embodiments of thepresent invention may be selected from a very broad range of structuraltypes. Preferred moieties include C₂-C₂₀ alkyl, aryl, or dialkylarylstructures where alkyl and25 aryl are defined as above. Linker moietiesmay be conveniently joined to monomers R1 and R2 through functionalgroups such as ethers, amides, ureas, carbamates, and esters; or throughalkyl-alkyl, alkyl-aryl, or aryl-aryl carbon-carbon bonds. Furthermore,linker moieties may be optimized (e.g., by modification of chain lengthand/or substituents) to enhance pharmacokinetic properties of themultimerizing agent. Holt et al. (WO 9606097) and Kathryn et al. (J.Steroid Biochem. Molec. Biol., 49: 139-152) describe a number of linkermoieties that can be used to construct the hybrid ligands of the instantinvention (R1-Y-R2), the contents of these references are incorporatedby reference herein.

[0320] In other embodiments, linker sequences are specifically designedso that increased solubility and enhanced permeability results. This isimportant since the components of the hybrid molecule, R1 and R2, areorganic molecules with potentially low water solubility. By linking twosmall molecules, the molecular weight is obviously increased,potentially further decreasing the water solubility and diffusioncoefficient. By designing a linker that increases solubility andenhances permeability of the hybrid, the available R1-Y-R2 hybrid insolution and ultimately inside the cell is effectively increased, sothat significantly higher sensitivity of the whole system can beachieved. In one embodiment, from 2 to 25 repeats of polyethylenglycol(PEG) groups of the general formula CH₂XCH₂ can be used, wherein Xrepresents O, S, SO, or SO₂. The number of repeats is preferably in therange of 3-25, 5-25, 9-25, 2-15, 3-15, 5-15 or 9-15, and morespecifically is preferably 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 2. In amost preferred embodiment, three polyethylenglycol groups are used aslinker which offer significantly better solubility and membranepermeability (see example 7 and GPC 285937 below). In other cases wherean even more strongly increased solubility and/or membrane permeabilityis desired, five repeats may be used. Furthermore, it should beunderstood that modifications of the side-chains of the linker can beeasily achieved without adversely affecting the solubility, membranepermeability, and/or overall biological activity of the compound, andtherefore, such derivative linker sequence units are also within thescope of the invention.

[0321] Below are presented several examples for hybrid molecules asenvisaged by the present invention. (CH₂XCH₂)_(n)-groups, wherein Xrepresents 0, n=3 or 5, were employed for these examples, withoutlimitation. Increasing the length of the linker sequence appears toincrease the effectiveness of the compound in at least some three-hybridassays, which is most likely due to the increased solubility or membranepermeability or flexibility of the molecule, or a combination thereof.For example, the n-octanol-water partition coefficient (clogP) of thecompound Mtx-mdbt-Dex is predicted by structure based calculations usingthe program Kowwin (Syracuse Research Corporation) to be 3.62, and it'swater solubility to lie in the range of 0.00035 mg/l, while clogP forGPC 285937, identical with Mtx-mdbt-Dex except for the replaced linker,is estimated by the same method to be −1.71, and its solubility as 0.13mg/l, corresponding to a factor of approximately 300 in increasedsolubility.

[0322] Structure of Mtx-mdbt-Dex (R1=Methothrexate, R2=Dexamethasone,Y=metadibenzothioester)

[0323] Structure of GPC 285937 (R1=Methothrexate, R2=Dexamethasone,Y═(CH₂—CH₂—O)₃)

[0324] 4-(N-{2-[2-(2-{2-[((2S, 11S,15S,17S, 1R, 13R,14R)-1-fluoro-14,17-dihydroxy-2,13,15-trimethyl-5-oxotetracyclo[8.7.0.0<2,7>0.0<11,15>]heptadeca-3,6-dien-14-yl)carbonylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]butanoicacid

[0325] Structure of GPC 285985 (R1=Methothrexate, Y═(CH₂—CH₂—O)₃, R2 isan active CDK2-inhibitor)

[0326]2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino[phenyl)carbonylam[no]-4-(N-{2-[2-(2-{2-[2-methyl-2-(4-{[3-(methylethyl)-4-oxo-1-(2,4,6-trichlorophenyl)(5-hydropyrazolo[5,4-d]pyrimidin-6-yl)]methyl}phenoxy)propanoylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)butanoicacid

[0327] Structure of GPC 285993 (R1=Methothrexate, Y═(CH₂—CH₂—O)₃, R2 isinactive as CDK2-inhibitor)

[0328]2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]-4-{N-[2-(2-{2-[2-(2-{3-(4-hydroxyphenyl)-5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-2-yl}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoicacid

[0329] Structure of GPC 286004 (R1=Methothrexate, Y═(CH₂—CH₂—O)₃, R2 isan active CDK2-inhibitor)

[0330]2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]-4-(N-{2-[2-(2-{2-[2-(4-{5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)butanoicacid

[0331] Structure of GPC 286026 (R1=Methothrexate, Y═(CH₂—CH₂—O)₅, R2 isan active CDK2-inhibitor)

[0332]2-[(4-{[(2,4-diaminoptedin-6-yl)methyl]methylamino]phenyl)carbonylamino]-4-{N-[2-(2-{2-[2-(2-{2-[2-(4-{5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoicacid

[0333] In a preferred embodiment, more than one hybrid small molecule isemployed for screening, wherein R1 and/or R2 are linked via the samelinker sequence but using different reaction groups in such a way sothat the relative orientation of R1 and R2 can be adjusted. This isuseful in optimization of an effective compound ligand since certainorientations might overcome or at least alleviate potential sterichinderances that serve to weaken the interaction between the ligand andits protein binding partner.

[0334] The structures of the hybrid small molecules shown above are byno means to be understood as limiting the scope of the presentinvention.

[0335] 5.2. High Affinity Ligands/Ligand Binding Proteins

[0336] According to the invention, two pairs of polypeptide/smallchemical compound interactions have to be present for the three-hybridsystem to activate a reporter system. One pair of interaction is betweena known ligand and its known polypeptide binding partner. Thisessentially serves as an “adaptor” to create a R2::P2 interactioninterface, and to provide the necessary second element of the reportersystem, RS2. Therefore, the stronger the P1::R1 interaction, the betterthe overall performance of the system.

[0337] There are at least two categories of P1::R1 interactionsavailable for this purpose: covalent and non-covalent interactions.Covalent interactions are almost always stronger. For example, certainenzymes and their suicide inhibitors or suicide substrates can beexploited to constitute such covalent interaction pairs. Suicideinhibitors or suicide substrates bind to their prospective enzymes withhigh specificity and affinity. Once bound, a chemical reaction occurs,physically linking the inhibitor/substrate to the enzyme, usually at itsactive site, thereby irreversibly inactivates the enzyme. If such enzymeis used as P1 and its suicide inhibitor/substrate used as R1 in thethree-hybrid system, a covalent link between P1-R1 can be established.For example, beta-lactamase may covalently bind suicide inhibitors suchas beta-lactam antibiotics. However, there are only limited selectionsof these enzyme—substrate/inhibitor pairs, particularly when thesubstrate/inhibitor needs to be connected to another small compound R2via a linker yet still retains solubility and membrane permeability invivo.

[0338] On the other hand, non-covalent P1::R1 interactions are moreversatile. There are many known high affinity ligand-receptorinteractions that can be employed in the three-hybrid system. Forexample, FK506 and FKBP (FK506 Binding Protein), FK506 and Rapamycin,biotin and streptavidin, DHFR and methotroxate (Mtx), glucocorticoidreceptor and Dexamethasone (Dex), etc, represent binding pairs withaffinities high enough to be potentially suitable as ligand receptorbinding pairs. The DHFR-Mtx interaction offers pM affinity, andtherefore is much better than FK506-FKBP interaction.

[0339] Any of a number of ligand/ligand binding protein pairs known inthe art may be utilized. For example, the steroid molecule,dexamethasone, which binds the glucocorticoid receptor with highaffinity may be employed. Dexamethasone is modular in nature; it can becovalently linked to another small molecule such as biotin withoutlosing its affinity for the glucocorticoid receptor- The use of steroidssuch as dexamethasone is advantageous in that these molecules are highlymembrane permeable and are small in size. The method of the inventionmay utilize other steroid molecules as well as small molecules otherthan steroids as ligand R1. Other ligands such as cyclosporin (M.W.1200) may also be used where the target or receptor to which the ligandis bound has been identified in the art. As another example, the smallmolecule FK506 (M.W. 850) which binds an FK binding protein (FKBP), andmodified derivatives of FK506 (i.e. “bump” modified compounds) whichbind to modified FK binding proteins (i.e. FKBP mutants which compensatefor-such “bump” modifications) are also adaptable for use asligand/ligand-binding proteins of the invention (see e.g. U.S. Pat. No.6,054,436, the contents of which are incorporated herein by reference).

[0340] Table 1 provides a list of ligands and ligand-binding pairs whichare known in the art and adaptable to the compositions and methods ofthe invention. Particularly preferred ligand/ligand-binding proteinpairs have strong binding affinities as reflected in low dissociationconstants (e.g., methotrexate/DHFR at 52 pM; ordexamethasone/glucocorticoid receptor at 86 nM). TABLE 1 List of SomeHigh Affinity Ligand/Ligand Binding Proteins Molecular Ligand weight (D)Ligand Binding Protein Affinity Biotin (244) Avidin 80 fM Ni  (59) 6 XHis 0.8 μM Rapamycin (914) FKB12 12 μM FK506 (804) FKB12 12 μMMethotrexate (454) DHFR 52 pM Tetracyclin (444) Tet-R 24 nMDexamathasone (392) Glucocorticoid receptor 86 nM Glutathione (307)Glutathione-S-Transferase 24 μM Maltose (342) Maltose Binding Protein 40nM Novobiotin (612) GyrB 123 μM

[0341] In general, virtually any ligand/ligand-binding protein pair withsufficient affinity may be adapted to the compositions and methods ofthe invention. Particularly preferred embodiments utilize ligand bindingproteins which are known to function efficiently intracellularly. Forexample, steroid receptors occur intracellularly and bind with highaffinities to their cognate steroid hormones under intracellularphysiological conditions. Examples of such steroid receptors include thehuman estrogen receptor (e.g. GenBank Accession No. NM_(—)000125), whichis found in estrogen-sensitive animal cells, and human glucocorticoidreceptor protein (e.g. GenBank Accession No. NM_(—)004491), which isfound in cells responsive to glucocorticoid hormones-Other steroids withsuitable receptors for use in the invention include testosterone,progesterone, and cortisone.

[0342] It should be understood that the above mentioned ligands shallalso include those derivatives and equivalents that share closestructural relationship to those ligands. To illustrate, Mtx only usesits 2,4-diaminopteridine double-ring structure to bind DHFR. Therefore,2,4-diaminopteridine shall be considered a derivative of Mtx that isalso within the scope of the invention. A “derivative” generally sharesthe effective moiety with the original compound but may also have othernon-essential structural elements for a given activity.

[0343] Still other preferred ligands for use in the invention are knownin the art and may be adapted to the methods and compositions of theinvention by skilled artisan without undue experimentation. For example,other preferred ligands which could be adapted to the invention includefat-soluble vitamins with cognate receptors such as Vitamin D and itsvarious forms such as D₁, D₂ (9,10-secoergosta-5, 7, 10 (19),22-tetraen-3-ol), D₃ (9,10-secocholeta-5, 7, 10(19)-trien-3-ol) and D₄(9, 10-secoergosta-5, 7, 10(19)-trien-3-ol). Vitamin D₃ binds withaffinity to the human nuclear vitamin D receptor protein (e.g. GenBankAccession No. NM_(—)000376; see also Haussler et al. (1995) Bone 17:33S-38S) and this ligand/ligand-binding protein pair may be adapted tothe invention. Still other ligands with cognate ligand-binding proteinsthat may be adapted to the invention include thyroid hormone andretinoic acid. DeWolf and Brett ((2000) Pharmacol Rev. 52: 207-36)provides a summary of many useful ligand-binding proteins with cognateligands including: biotin-binding proteins, lipid-binding protein,periplasmic binding proteins, lectins, serum albumins, immunoglobulins,various inactivated enzymes, insect pheromone binding proteins,odorant-binding proteins, immunosuppressant-binding proteins, phosphate-and sulfate-binding protein.

[0344] In addition, steroid, retinoic acid, beta-lactam antibiotic,cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivatives,rapamycin, tetracycline, methotrexate, 2,4-diaminopteridine, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, niche, cyelosporin and theirnatural or synthesized binding partners are all possible for use in theinstant invention as a component of the above described high affinityligand/ligand binding pair. In all those compounds mentioned above, itshould be understood that basically equivalent compounds with only minorstructural variations can also be used.

[0345] On the other hand, a user-specified second ligand need to belinked to the above-described ligand to form a compound ligand. At leastthe following chemical groups and those basically equivalent compoundswith only minor structural variations can be used as such user-specifiedligands: a peptide, a nucleic acid, a carbohydrate, a polysaccharide, alipid, a prostaglandin, an acyl halide, an alcohol, an aldehyde, analkane, an alkene, an alkyne, an alkyl, an alkyl halide, an alkaloid, anamine, an aromatic hydrocarbon, a sulfonate ester, a carboxylate acid,an aryl halide, an ester, a phenol, an ether, a nitrile, a carboxylicacid anhydride, an amide, a quaternary ammonium salt, an imine, anenamine, an amine oxide, a cyanohydrin, an organocadmium, an aldol, anorganometallic, an aromatic hydrocarbon, a nucleoside, a nucleotide. Forexample, in a recent publication (U.S. Pat. No. 6,326,155), a method isdescribed that aids in selecting a ligand for a given target molecule.

[0346] 6. Libraries and Screening Methods

[0347] 6.1 Variegated Peptide Display

[0348] One aspect of the invention provides a method to identifypolypeptides that bind to a given small molecule/chemical compound. Thepolypeptides are usually provided in the form of a variegated library,which can contain different number of members, preferably from 2 to 10members, or 10 to 500 members, 500 to 10,000 members or more than 10,000members. The library can be a nucleic acid library (mRNA, cDNA, genomicDNA, EST, YAC, p1 clones, BAC/PAC libraries, etc.) which encodespolypeptides. Depending on the specific embodiments of the screens used(for example, split-ubiquitin based hybrid system or transcription basedyeast hybrid system), the nucleic acid library is usually constructed invectors suitable for the chosen embodiment, using art-recognizedtechniques.

[0349] The variegated peptide libraries of the subject method can begenerated by any of a number of methods, and, though not limited by,preferably exploit recent trends in the preparation of chemicallibraries. The library can be prepared, for example, by either syntheticor biosynthetic approaches. As used herein, “variegated” refers to thefact that a population of peptides is characterized by having a peptidesequence which differ from one member of the library to the next. Forexample, in a given peptide library of N amino acids in length, thetotal number of different peptide sequences in the library is given bythe product of (X₁*X₂* . . . X_(i)), where each X_(i) represents thenumber of different amino acid residues occurring at position X of thepeptide. In a preferred embodiment of the present invention, the peptidedisplay collectively produces a peptide library including at least 96 to10⁷ different peptides, so that diverse peptides may be simultaneouslyassayed for the ability to interact with the small molecule/chemicalcompound.

[0350] The polypeptide libraries can be prescreened for interactionswith the small molecule/chemical compound, for example using a phagedisplay method. Peptide libraries are systems which simultaneouslydisplay, in a form which permits interaction with a target molecule, ahighly diverse and numerous collection of peptides. These peptides maybe presented in solution (Houghten (1992) Biotechniques 13:412-421), oron beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (LadnerU.S. Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl AcadSci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990)Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol.222:301-310; and Ladner U.S. Pat. No. 5,223,409).

[0351] In one embodiment, the peptide library is derived to express acombinatorial library of peptides which are not based on any knownsequence, nor derived from cDNA. That is, the sequences of the libraryare largely random. It will be evident that the peptides of the librarymay range in size from dipeptides to large proteins.

[0352] In another embodiment, the peptide library is derived to expressa combinatorial library of peptides which are based at least in part ona known polypeptide sequence or a portion thereof (not a cDNA library).That is, the sequences of the library is semi-random, being derived bycombinatorial mutagenesis of a known sequence(s). See, for example,Ladner et al. PCT publication WO 9002909; Garrard et al., PCTpublication WO 9209690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461. Accordingly, polypeptide(s) which are known ligands for atarget molecule can be mutagenized by standard techniques to derive avariegated library of polypeptide sequences which can further bescreened for binding partners including agonists and/or antagonists.

[0353] In still another embodiment, the combinatorial polypeptides areproduced from a cDNA library, a genomic DNA library. The source of DNAcan be of human, non-human mammalian, fish, amphibium, insect, worm,yeast, plant, or bacteria.

[0354] Depending on size, the combinatorial peptides of the library canbe generated as is, or can be incorporated into larger fusion proteins,such as library-reporter system fusions. The fusion protein may alsoprovide, for example, stability against degradation or denaturation, aswell as a secretion signal if secreted, or the reporter functionnecessary for screens. In an exemplary embodiment, the polypeptidelibrary is provided as part of thioredoxin fusion proteins (see, forexample, U.S. Pat. Nos. 5,270,181 and 5,292,646; and PCT publicationWO9402502). The combinatorial peptide can be attached on the terminus ofthe thioredoxin protein, or, for short peptide libraries, inserted intothe so-called active loop. In another preferred embodiment, the fusionprotein library can be provided as a fusion to either the Cub or Nuxdomain of the split ubiquitin sensor proteins (see below). In anotherpreferred embodiment, the fusion protein library can be provided as afusion to either the DNA binding domain or the transcription activationdomain of the transcription based yeast three-hybrid system.

[0355] In preferred embodiments, the combinatorial polypeptides are inthe range of 3-1000 amino acids in length, more preferably at least5-500, and even more preferably at least 3-100, 5-50, 10, 13, 15, 20 or25 amino acid residues in length. Preferably, the polypeptides of thelibrary are of uniform length. It will be understood that the length ofthe combinatorial peptide does not reflect any extraneous sequenceswhich may be present in order to facilitate expression, e.g., such assignal sequences or invariant portions of a fusion protein.

[0356] Regardless of the nature of the peptide libraries, the samepeptide libraries can also be provided as nucleic acid librariesencoding such peptide libraries. These nucleic acid libraries can beprovided in suitable vectors for expression in various systems,including, but are not limited to mammalian, insect, yeast and bacteriaexpression systems. A skilled artisan shall be able to determine theappropriate vectors to use for various expression systems.

[0357] 6.1.1 Biosynthetic Peptide Libraries

[0358] The harnessing of biological systems for the generation ofpeptide diversity is now a well established technique which can beexploited to generate the peptide libraries of the subject method. Thesource of diversity is the combinatorial chemical synthesis of mixturesof oligonucleotides. Oligonucleotide synthesis is a well-characterizedchemistry that allows tight control of the composition of the mixturescreated. Degenerate DNA sequences produced are subsequently placed intoan appropriate genetic context for expression as peptides.

[0359] There are two principal ways in which to prepare the requireddegenerate mixture. In one method, the DNAs are synthesized a base at atime. When variation is desired at a base position dictated by thegenetic code a suitable mixture of nucleotides is reacted with thenascent DNA, rather than the pure nucleotide reagent of conventionalpolynucleotide synthesis. The second method provides more exact controlover the amino acid variation. First, trinucleotide reagents areprepared, each trinucleotide being a codon of one (and only one) of theamino acids to be featured in the peptide library. When a particularvariable residue is to be synthesized, a mixture is made of theappropriate trinucleotides and reacted with the nascent DNA. Once thenecessary “degenerate” DNA is complete, it must be joined with the DNAsequences necessary to assure the expression of the peptide, asdiscussed in more detail below, and the complete DNA construct must beintroduced into the cell.

[0360] Whatever the method may be for generating diversity at the codonlevel, chemical synthesis of a degenerate gene sequence can be carriedout in an automatic DNA synthesizer, and the synthetic genes can then beligated into an appropriate gene or vector for expression. The purposeof a degenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential test peptide sequences.The synthesis of degenerate oligonucleotides is well known in the art(see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al.(1981) Recombinant DNA, Proc 3^(rd) Cleveland Sympos. Macromolecules,ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984)Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198 :1056; Ikeet al. (1983) Nucleic Acid Res. 11:477. Such techniques have beenemployed in the directed evolution of other proteins (see, for example,Scott et al. (1990) Science 249 :386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249 : 404-406; Cwirla et al.(1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

[0361] Because the number of different peptides one can create by thiscombination approach can be huge, and because the expectation is thatpeptides with the appropriate structural characteristics to serve asligands for a given target protein will be rare in the total populationof the library, the need for methods capable of conveniently screeninglarge numbers of clones is apparent. Several strategies for selectingpeptide ligands from the library have been described in the art and areapplicable to certain embodiments of the present method.

[0362] The number of possible peptides for a given library may, incertain instances, exceed 10¹². To sample as many combinations aspossible depends, in part, on the ability to recover large numbers oftransformants. For phage with plasmid-like forms (as filamentous phage),electrotransformation provides an efficiency comparable to that ofphage-transfection with in vitro packaging, in addition to a very highcapacity for DNA input. This allows large amounts of vector DNA to beused to obtain very large numbers of transformants. The method describedby Dower et al. (1988) Nucleic Acids Res., 16:6127-6145, for example,may be used to transform fd-tet derived recombinants at the rate ofabout 10⁷ transformants/μg of ligated vector into E. coli (such asstrain MC1061), and libraries may be constructed in fd-tet B1 of up toabout 3×10⁸ members or more. Increasing DNA input and makingmodifications to the cloning protocol within the ability of the skilledartisan may produce increases of greater than about 10-fold in therecovery of transformants, providing libraries of up to 10¹⁰ or morerecombinants.

[0363] 6.1.2 Synthetic Peptide Libraries

[0364] In contrast to the recombinant methods, in vitro chemicalsynthesis provides a method for generating libraries of compounds,without the use of living organisms, that can be screened for ability tobind to a target molecule. Although in vitro methods have been used forquite some time in the pharmaceutical industry to identify potentialdrugs, recently developed methods have focused on rapidly andefficiently generating and screening large numbers of compounds and areparticularly amenable to generating peptide libraries for use in thesubject method.

[0365] One particularly useful features of the synthetic peptide libraryis that it can be used to supply libraries of R2 to be coupled to R1-Y,in order to make the hybrid ligand. This can be used to screen for asynthetic polypeptide that can bind a user-specified polypeptide. Forexample, the synthetic polypeptide can be a potential peptide inhibitorof a user-specified enzyme or transcription factor, etc. Such screenscan be a prescreen of large number of random polypeptides in an in vitrohigh-throughput setting, so that primary positive peptides can beselected, and its variants encoded by a nucleic acid library furtherscreened in an in vivo embodiment.

[0366] Another use for the synthetic peptide library is to generatelibraries of short peptide linkers to be inserted between R1 and R2ligands. This is particularly useful since an optimal linker sequencemay be generated for a particular R1-R2 pair, so that the final hybridligand may possess the optimal chemical and/or structuralcharacteristics such as solubility, membrane permeability, etc.

[0367] Both uses require coupling of a synthetic polypeptide, usingknowledge well-known in the art (such as the ones described below orelsewhere), to another molecule (linker Y or ligands R1 and R2), whichmay be peptide or non-peptide in nature.

[0368] The various approaches to simultaneous preparation and analysisof large numbers of synthetic peptides (herein “multiple peptidesynthesis” or “MPS”) each rely on the fundamental concept of synthesison a solid support introduced by Merrifield in 1963 (Merrifield, R. B.(1963) J Am Chem Soc 85:2149-2154; and references cited in section Iabove). Generally, these techniques are not dependent on the protectinggroup or activation chemistry employed, although most workers todayavoid Merrifield's original tBoc/Bz1 strategy in favor of the more mildFmoc/tBu chemistry and efficient hydroxybenzotriazole-based couplingagents. Many types of solid matrices have been successfully used in MPS,and yields of individual peptides synthesized vary widely with thetechnique adopted (e.g., nanomoles to millimoles).

[0369] 6.1.2.1 Multipin Synthesis

[0370] One form that the peptide library of the subject method can takeis the multipin library format. Briefly, Geysen and co-workers (Geysenet al. (1984) PNAS 81:3998-4002) introduced a method for generatingpeptide by a parallel synthesis on polyacrylic acid-grated polyethylenepins arrayed in the microtitre plate format. In the originalexperiments, about 50 mmol of a single peptide sequence was covalentlylinked to the spherical head of each pin, and interactions of eachpeptide with receptor or antibody could be determined in a directbinding assay. The Geysen technique can be used to synthesize and screenthousands of peptides per week using the multipin method, and thetethered peptides may be reused in many assays. In subsequent work, thelevel of peptide loading on individual pins has been increased to asmuch as 2 μmol/pin by grafting greater amounts of functionalizedacrylate derivatives to detachable pin heads, and the size of thepeptide library has been increased (Valerio et al. (1993) Int J PeptProtein Res 42:1-9). Appropriate linker moieties have also been appendedto the pins so that the peptides may be cleaved from the supports aftersynthesis for assessment of purity and evaluation in competition bindingor functional bioassays (Bray et al. (1990) Tetrahedron Lett31:5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray etal. (1991) Tetrahedron Lett 32:6163-6166).

[0371] More recent applications of the multipin method of MPS have takenadvantage of the cleavable linker strategy to prepare soluble peptides(Maeji et al. (1990) J Immunol Methods 134:23-33; Gammon et al. (1991) JExp Med 173:609-617; Mutch et al. (1991) Pept Res 4:132-137).

[0372] 6.1.2.2 Divide-Couple-Recombine

[0373] In yet another embodiment, a variegated library of peptides canprovide on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy is introduced intothe library, the beads are divided into as many separate groups tocorrespond to the number of different amino acid residues to be addedthat position, the different residues coupled in separate reactions, andthe beads recombined into one pool for the next step.

[0374] In one embodiment, the divide-couple-recombine strategy can becarried out using the so-called “tea bag” MPS method first developed byHoughten, peptide synthesis occurs on resin that is sealed inside porouspolypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Aminoacids are coupled to the resins by placing the bags in solutions of theappropriate individual activated monomers, while all common steps suchas resin washing and amino group deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single peptide sequence, and the peptides may beliberated from the resins using a multiple cleavage apparatus (Houghtenet al. (1986) Int J Pept Protein Res 27:673-678). This technique offersadvantages of considerable synthetic flexibility and has been partiallyautomated (Beck-Sickinger et al. (1991) Pept Res 4:88-94). Moreover,soluble peptides of greater than 15 amino acids in length can beproduced in sufficient quantities (>0.5 mmol) for purification andcomplete characterization if desired.

[0375] Multiple peptide synthesis using the tea-bag approach is usefulfor the production of a peptide library, albeit of limited size, forscreening the present method, as is illustrated by its use in a range ofmolecular recognition problems including antibody epitope analysis(Houghten et al. (1986) PNAS 82:5131-5135), peptide hormonestructure-function studies (Beck-Sickinger et al. (1990) Int J PeptProtein Res 36:522-530; Beck-Sickinger et al. (1990) Eur J Biochem194:449-456), and protein conformational mapping (Zimmerman et al.(1991) Eur J Biochem 200:519-528).

[0376] An exemplary synthesis of a set of mixed peptides havingequimolar amounts of the twenty natural amino acid residues is asfollows. Aliquots of five grams (4.65 mmols) of p-methylbenzhydrylaminehydrochloride resin (MBHA) are placed into twenty porous polypropylenebags. These bags are placed into a common container and washed with 1.0liter of CH₂Cl₂ three times (three minutes each time), then again washedthree times (three minutes each time) with 1.0 liter of 5 percentDIEA/CH₂Cl₂ (DIEA=diisopropylethylamine; CH₂Cl₂=DCM). The bags are thenrinsed with DCM and placed into separate reaction vessels eachcontaining 50 ml (0.56 M) of the respective t-BOC-amino acid/DCM.N,N-Diisopropylcarbodiimide (DIPCDI; 25 ml; 1.12 M) is added to eachcontainer, as a coupling agent. Twenty amino acid derivatives areseparately coupled to the resin in 50/50 (v/v) DMF/DCM. After one hourof vigorous shaking, Gisen's picric acid test (Gisen (1972) Anal. Chem.Acta 58:248-249) is performed to determine the completeness of thecoupling reaction. On confirming completeness of reaction, all of theresin packets are then washed with 1.5 liters of DMF and washed two moretimes with 1.5 liters of CH₂Cl2. After rinsing, the resins are removedfrom their separate packets and admixed together to form a pool in acommon bag. The resulting resin mixture is then dried and weighed,divided again into 20 equal portions (aliquots), and placed into 20further polypropylene bags (enclosed).

[0377] In a common reaction vessel the following steps are carried out:(1) deprotection is carried out on the enclosed aliquots for thirtyminutes with 1.5 liters of 55% TFA/DCM; and 2) neutralization is carriedout with three washes of 1.5 liters each of 5% DIEA/DCM. Each bag isplaced in a separate solution of activated t-BOC-amino acid derivativeand the coupling reaction carried out to completion as before. Allcoupling reactions are monitored using the above quantitative picricacid assay.

[0378] Next, the bags are opened and the resulting t-BOC-protecteddipeptide resins are mixed together to form a pool, aliquots are madefrom the pool, the aliquots are enclosed, deprotected and furtherreactions are carried out. This process can be repeated any number oftimes yielding at each step an equimolar representation of the desirednumber of amino acid residues in the peptide chain. The principalprocess steps are conveniently referred to as a divide-couple-recombinesynthesis.

[0379] After a desired number of such couplings and mixtures are carriedout, the polypropylene bags are kept separated to here provide thetwenty sets having the amino-terminal residue as the single,predetermined residue, with, for example, positions 2-4 being occupiedby equimolar amounts of the twenty residues. To prepare sets having thesingle, predetermined amino acid residue at other than theamino-terminus, the contents of the bags are not mixed after adding aresidue at the desired, predetermined position. Rather, the contents ofeach of the twenty bags are separated into 20 aliquots, deprotected andthen separately reacted with the twenty amino acid derivatives. Thecontents of each set of twenty bags thus produced are thereafter mixedand treated as before-described until the desired oligopeptide length isachieved.

[0380] 6.1.2.3 Multiple Peptide Synthesis through Coupling of Amino AcidMixtures

[0381] Simultaneous coupling of mixtures of activated amino acids to asingle resin support has been used as a multiple peptide synthesisstrategy on several occasions (Geysen et al. (1986) Mol Immunol 23:709-715; Tjoeng et al. (1990) Int J Pept Protein Res 35 :141-146;Rutter et al. (1991) U.S. Pat. No. 5,010,175; Birkett et al. (1991) AnalBiochem 196:137-143; Petithory et al. (1991) PNAS 88:11510-11514) andcan have applications in the subject method. For example, four to sevenanalogs of the magainin 2 and angiotensinogen peptides were successfullysynthesized and resolved in one HPLC purification after coupling amixture of amino acids at a single position in each sequence (Tjoeng etal. (1990) Int J Pept Protein Res 35 :141-146). This approach has alsobeen used to prepare degenerate peptide mixtures for defining thesubstrate specificity of endoproteolytic enzymes (Birkett et al. (1991)Anal Biochem 196:137-143; Petithory et al. (1991) PNAS 88:11510-11514).In these experiments a series of amino acids was substituted at a singleposition within the substrate sequence. After proteolysis, Edmandegradation was used to quantitate the yield of each amino acidcomponent in the hydrolysis product and hence to evaluate the relativekcat/Kn values for each substrate in the mixture.

[0382] However, it is noted that the operational simplicity ofsynthesizing many peptides by coupling monomer mixtures is offset by thedifficulty in controlling the composition of the products. The productdistribution reflects the individual; rate constants for the competingcoupling reactions, with activated derivatives of sterically hinderedresidues such as valine or isoleucine adding at a significantly slowerrate than glycine or alanine for example. The nature of the resin-boundcomponent of the acylation reaction also influences the addition rate,and the relative rate constants for the formation of 400 dipeptides formthe 20 genetically coded amino acids have been determined by Rutter andSanti (Rutter et al. (1991) U.S. Pat. No. 5,010,175). These reactionrates can be used to guide the selection of appropriate relativeconcentrations of amino acids in the mixture to favor more closelyequimolar coupling yields.

[0383] 6.1.2.4 Multiple Peptide Synthesis on Nontraditional SolidSupports

[0384] The search for innovative methods of multiple peptide synthesishas led to the investigation of alternative polymeric supports to thepolystyrene-divinylbenzene matrix originally popularized by Merrifield.Cellulose, either in the form of paper disks (Blankemeyer-Menge et al.(1988) Tetrahedron Lett 29-5871-5874; Frank et al. (1988) Tetrahedron 44:6031-6040; Eichler et al. (1989) Collect Czech Chem Commun54:1746-1752; Frank, R. (1993) Bioorg Med Chem Lett 3:425-430) or cottonfragments (Eichler et al. (1991) Pept Res 4 :296-307; Schmidt et al.(1993) Bioorg Med Chem Lett 3:441-446) has been successfullyfunctionalized for peptide synthesis. Typical loadings attained withcellulose paper range from 1 to 3 mmol/cm², and HPLC analysis ofmaterial cleaved from these supports indicates a reasonable quality forthe synthesized peptides. Alternatively, peptides may be synthesized oncellulose sheets via non-cleavable linkers and then used in ELISA-basedbinding studies (Frank, R. (1992) Tetrahedron 48:9217-9232). The porous,polar nature of this support may help suppress unwanted nonspecificprotein binding effects. By controlling the volume of activated aminoacids and other reagents spotted on the paper, the number of peptidessynthesized at discrete locations on the support can be readily varied.In one convenient configuration spots are made in an 8×12 microtiterplate format. Frank has used this technique to map the dominant epitopesof an antiserum raised against a human cytomegalovirus protein,following the overlapping peptide screening (Pepscan) strategy of Geysen(Frank, R. (1992) Tetrahedron 48:9217-9232). Other membrane-likesupports that may be used for multiple solid-phase synthesis includepolystyrene-grafted polyethylene films (Berg et al. (1989) J Am Chem Soc111:8024-8026).

[0385] 6.1.2.5 Combinatorial Libraries by Light-Directed, SpatiallyAddressable Parallel Chemical Synthesis

[0386] A scheme of combinatorial synthesis in which the identity of acompound is given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S.P.A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The technique combines two well-developedtechnologies: solid-phase peptide synthesis chemistry andphotolithography. The high coupling yields of Merrifield chemistry allowefficient peptide synthesis, and the spatial resolution ofphotolithography affords miniaturization. The merging of these twotechnologies is done through the use of photolabile amino protectinggroups in the Merrifield synthetic procedure.

[0387] The key points of this technology are illustrated in Gallop etal. (1994) J Med Chem 37:1233-1251. A synthesis substrate is preparedfor amino acid coupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers. Light is usedto selectively activate a specified region of the synthesis support forcoupling. Removal of the photolabile protecting groups by lights(deprotection) results in activation of selected areas. Afteractivation, the first of a set of amino acids, each bearing aphotolabile protecting group on the amino terminus, is exposed to theentire surface. Amino acid coupling only occurs in regions that wereaddressed by light in the preceding step. The solution of amino acid isremoved, and the substrate is again illuminated through a second mask,activating a different region for reaction with a second protectedbuilding block. The pattern of masks and the sequence of reactantsdefine the products and their locations. Since this process utilizesphotolithography techniques, the number of compounds that can besynthesized is limited only by the number of synthesis sites that can beaddressed with appropriate resolution. The position of each compound isprecisely known; hence, its interactions with other molecules can bedirectly assessed. The target protein can be labeled with a fluorescentreporter group to facilitate the identification of specific interactionswith individual members of the matrix.

[0388] In a light-directed chemical synthesis, the products depend onthe pattern of illumination and on the order of addition of reactants.By varying the lithographic patterns, many different sets of testpeptides can be synthesized in the same number of steps; this leads tothe generated of many different masking strategies.

[0389] 6.1.2.6 Encoded Combinatorial Libraries

[0390] In yet another embodiment, the subject method utilizes a peptidelibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries above, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. Two forms of encoding have beenreported: encoding with sequenceable bio-oligomers (e.g.,oligonucleotides and peptides), and binary encoding withnon-sequenceable tags.

[0391] 6.1.2.6.1 Tagging with Sequenceable Bio-Oligomers

[0392] The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 77 (=823,543) peptides composed of all combinationsof Arg, Gln, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH2groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following the such techniques, the peptide librariescan be derived for use in the subject method and screened using theD-enantiomer of the target protein.

[0393] It is noted that an alternative approach useful for generatingnucleotide-encoded synthetic peptide libraries employs a branched linkercontaining selectively protected OH and NH2 groups (Nielsen et al.(1993) J Am Chem Soc 115:9812-9813; and Nielsen et al. (1994) MethodsCompan Methods Enzymol 6:361-371). This approach requires that equimolarquantities of test peptide and tag co-exist, though this may be apotential complication in assessing biological activity, especially withnucleic acid based targets.

[0394] The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical liability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesison non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test peptidelibrary member for bioassay, in part (as described infra) because assaysemploying beads limit the choice of targets, and in part because thetags are potentially susceptible to biodegradation.

[0395] Peptides themselves have been employed as tagging molecules forcombinatorial libraries. Two exemplary approaches are described in theart, both of which employ branched linkers to solid phase upon whichcoding and ligand strands are alternately elaborated. In the firstapproach (Kerr J M et al. (1993) J Am Chem Soc 115:2529-2531),orthogonality in synthesis is achieved by employing acid-labileprotection for the coding strand and base-labile protection for theligand strand.

[0396] In an alternative approach (Nikolaiev et al. (1993) Pept Res6:161-170), branched linkers are employed so that the coding unit andthe test peptide are both attached to the same functional group on theresin. In one embodiment, a linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and ligand (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). Inanother embodiment, the linker can be placed so that the test peptidecan be selectively separated from the bead, leaving the code behind.This last construct is particularly valuable because it permitsscreening of the test peptide without potential interference, orbiodegradation, of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

[0397] It is noted that peptide tags are more resistant to decompositionduring ligand synthesis than are oligonucleotide tags, but they must beemployed in molar ratios nearly equal to those of the ligand on typical130 mm beads in order to be successfully sequenced. As witholigonucleotide encoding, the use of peptides as tags requires complexprotection/deprotection chemistries.

[0398] 6.1.2.6.2 Non-Sequenceable Tagging: Binary Encoding

[0399] An alternative form of encoding the test peptide library employsa set of non-sequenceable electrophoric tagging molecules that are usedas a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplarytags are haloaromatic alkyl ethers that are detectable as theirtetramethylsilyl ethers at less than femtomolar levels by electroncapture gas chromatography (ECGC). Variations in the length of the alkylchain, as well as the nature and position of the aromatic halidesubstituents, permit the synthesis of at least 40 such tags, which inprinciple can encode 240 (e.g., upwards of 1012) different molecules. Inthe original report (Ohlmeyer et al., supra) the tags were bound toabout 1% of the available amine groups of a peptide library via aphotocleavable O-nitrobenzyl linker. This approach is convenient whenpreparing combinatorial libraries of peptides or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, theligand is attached to the solid support via the photocleavable linkerand the tag is attached through a catechol ether linker via carbeneinsertion into the bead matrix (Nestler et al. (1994) J Org Chem59:4723-4724). This orthogonal attachment strategy permits the selectivedetachment of library members for bioassay in solution and subsequentdecoding by ECGC after oxidative detachment of the tag sets.

[0400] Binary encoding with electrophoric tags has been particularlyuseful in defining selective interactions of substrates with syntheticreceptors (Borchardt et al. (1994) J Am Chem Soc 116:373-374), and modelsystems for understanding the binding and catalysis of biomolecules.Even using detailed molecular modeling, the identification of theselectivity preferences for synthetic receptors has required the manualsynthesis of dozens of potential substrates. The use of encodedlibraries makes it possible to rapidly examine all the members of apotential binding set. The use of binary-encoded libraries has made thedetermination of binding selectivities so facile that structuralselectivity has been reported for four novel synthetic macrobicyclic andtricyclic receptors in a single communication (Wennemers et al. (1995) JOrg Chem 60:1108-1109; and Yoon et al. (1994) Tetrahedron Lett35:8557-8560) using the encoded library mentioned above. Similarfacility in defining specificity of interaction would be expected formany other biomolecules.

[0401] Although the several amide-linked libraries in the art employbinary encoding with the electrophoric tags attached to amine groups,attaching these tags directly to the bead matrix provides far greaterversatility in the structures that can be prepared in encodedcombinatorial libraries. Attached in this way, the tags and their linkerare nearly as unreactive as the bead matrix itself. Two binary-encodedcombinatorial libraries have been reported where the electrophoric tagsare attached directly to the solid phase (Ohlmeyer et al. (1995) PNAS92:6027-6031) and provide guidance for generating the subject peptidelibrary. Both libraries were constructed using an orthogonal attachmentstrategy in which the library member was linked to the solid support bya photolabile linker and the tags were attached through a linkercleavable only by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second, ligandsare partially detached and transferred to assay plates; third, abioassay identifies the active wells; fourth, the corresponding beadsare rearrayed singly into new microtiter plates; fifth, single activecompounds are identified; and sixth, the structures are decoded.

[0402] The above approach was employed in screening for carbonicanhydrase (CA) binding and identified compounds which exhibitednanomolar affinities for CA. Unlike sequenceable tagging, a large numberof structures can be rapidly decoded from binary-encoded libraries (asingle ECGC apparatus can decode 50 structures per day). Thus,binary-encoded libraries can be used for the rapid analysis ofstructure-activity relationships and optimization of both potency andselectivity of an active series. The synthesis and screening of largeunbiased binary encoded peptide libraries for lead identification,followed by preparation and analysis of smaller focused libraries forlead optimization, offers a particularly powerful approach to drugdiscovery using the subject method.

[0403] 6.1.3 Nucleic Acid Libraries

[0404] In another embodiment, the library is comprised of a variegatedpool of nucleic acids, e.g. single or double-stranded DNA or an RNA. Avariety of techniques are known in the art for generating screenablenucleic acid libraries which may be exploited in the present invention.The libraries that can be used with the instant invention includelibraries generated from: synthetic oligonucleotides, cDNA sequence,bacterial genomic DNA fragments, and eukaryotic genomic DNA fragments.

[0405] In particular, many of the techniques described above forsynthetic peptide libraries can be used to generate nucleic acidlibraries of a variety of formats. For example, divide-couple-recombinetechniques can be used in conjugation with standard nucleic acidsynthesis techniques to generate bead immobilized nucleic acidlibraries.

[0406] In another embodiment, solution libraries of nucleic acids can begenerated which rely on PCR techniques to amplify for sequencing thosenucleic acid molecules which selectively bind the screening target. Bysuch techniques, libraries approaching 10¹⁵ different nucleotidesequences have been generated in solution (see, for example, Bartel andSzostak (1993) Science 261: 1411-1418; Bock et al. (1992) Nature 355:564; Ellington et al. (1992) Nature 355: 850-852; and Oliphant et al.(1989) Mol Cell Biol 9: 2944-2949).

[0407] According to one embodiment of the subject method, the SELEX(systematic evolution of ligands by exponential enrichment) is employedwith the enantiomeric screening target. See, for example, Tuerk et al.(1990) Science 249:505-510 for a review of SELEX. Briefly, in the firststep of these experiments on a pool of variant nucleic acid sequences iscreated, e.g. as a random or semi-random library. In general, aninvariant 3′ and (optionally) 5′ primer sequence are provided for usewith PCR anchors or for permitting subcloning. The nucleic acid libraryis applied to screening a target, and nucleic acids which selectivelybind (or otherwise act on the target) are isolated from the pool. Theisolates are amplified by PCR and subcloned into, for example,phagemids. The phagemids are then transfected into bacterial cells, andindividual isolates can be obtained and the sequence of the nucleic acidcloned from the screening pool can be determined.

[0408] When RNA is the test ligand, the RNA library can be directlysynthesized by standard organic chemistry, or can be provided by invitro translation as described by Tuerk et al., supra. Likewise, RNAisolated by binding to the screening target can be reverse transcribedand the resulting cDNA subcloned and sequenced as above.

[0409] Isolation of mRNA for cDNA synthesis and isolation of genomicDNA, either of prokaryotic or eukaryotic origin, are well-known in theart of molecular biology. Many standard laboratory manuals such asCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 orlater editions), or Molecular Cloning: A Laboratory Manual, Cold SpringHarbor press (1989 or later editions) have detailed description of thesesubjects. In addition, many companies offer commercial kits specificallydesigned for such purposes.

[0410] 6.2 Small Molecule Libraries

[0411] Recent trends in the search for novel pharmacological agents havefocused on the preparation of chemical libraries. Peptide libraries aredescribed above. Nucleic acid libraries (including cDNA, genomic DNA andEST libraries) are well-known in the art. Saccharide libraries and theirsynthesis using combinatory chemistry have been described in WO 9816536and its related applications. However, the field of combinatorialchemistry has also provided large numbers of non-polymeric, smallorganic molecule libraries which can be employed in the subject method.

[0412] Exemplary combinatorial libraries include benzodiazepines,peptoids, biaryls and hydantoins. In general, the same techniquesdescribed above for the various formats of chemically synthesizedpeptide libraries may also be used to generate and (optionally) encodesynthetic non-peptide libraries.

[0413] 6.3 Selecting Compounds from the Library

[0414] As with the diversity contemplated for the compound library andform in which the compound library is provided, the subject method isenvisaged to identify hybrid ligands with the general formula of R1-Y-R2which interacts with a polypeptide screening target or to identifyinhibitors or antagonists of a certain interaction. In most embodiments,the screening programs test libraries of compounds/hybrid ligandssuitable for high throughput analysis in order to maximize the number ofcompounds surveyed in a given period of time. However, as a generalrule, the screening portion of the subject method involves contactingthe screening target with the compound library and isolating thosecompounds from the library which interact with the screening target orcausing a desired effect. Such interaction between the testcompound/hybrid ligands and the screening target may be detected, forexample, based on the change of status of any one of the suitablereporter system as described in section 3, or modulation of anenzymatic/catalytic activity of the screening target (for example, whenthe binding of a hybrid ligand for its potential dimerizable target istested). The efficacy of the test compounds can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound. Moreover, a control assay can alsobe performed to provide a baseline for comparison.

[0415] In one embodiment, the variegated compound library is subjectedto affinity enrichment in order to select for compounds which bind apreselected screening target. The term “affinity separation” or“affinity enrichment” includes, but is not limited to (1) affinitychromatography utilizing immobilizing screening targets, (2)precipitation using screening targets, (3) fluorescence activated cellsorting where the compound library is so amenable, (4) agglutination,and (5) plaque lifts. In each embodiment, the library of compounds areultimately separated based on the ability of a particular compound tobind a screening target of interest. See, for example, the Ladner et al.U.S. Pat. No. 5,223,409; the Kang et al. International Publication No.WO 9218619; the Dower et al. International Publication No. WO 9117271;the Winter et al. International Publication WO 9220791; the Markland etal. International Publication No. WO 9215679; the Breitling et al.International Publication WO 9301288; the McCafferty et al.International Publication No. WO 9201047; the Garrard et al.International Publication No. WO 9209690; and the Ladner et al.International Publication No. WO 9002809.

[0416] It will be apparent that, in addition to utilizing binding as theseparation criteria, compound libraries can be fractionated based onother activities of the target molecule, such as modulation of catalyticactivity or certain biochemical properties.

[0417] In one embodiment, binding between a chemical compound and atarget polypeptide can be measured by the activity of the reportersystem as described above. For example, if a ubiquitin based reportersystem is used for the detection, depending on the identity of theresidue Z (the first amino acid of the cleaved reporter moiety), thedetection could either be the presence of some activity of the reportermoiety (if Z is stabilizing amino acid like methionine) or the absenceof certain activity of the reporter moiety (if Z is a destabilizingnon-methionine amino acid). The activity to be detected could betranscription activity, fluorescence, enzymatic activity, or any otherbiological or biochemical activity described above. If a transcriptionbased reporter system is used for the detection, transcription activityof the reporter moiety can be monitored to screen for the compound orthe polypeptide binding to their target. Those skilled in the art willreadily appreciate and recognize other appropriate methods suitable forthose screens.

[0418] 7. Nucleic Acids

[0419] The invention provides nucleic acids, including certain genes andhomologs thereof, and portions thereof. Preferred nucleic acids have asequence at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%;70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, and morepreferably 85% homologous and more preferably 90% and more preferably95% and even more preferably at least 99% homologous with a nucleotidesequence of a particular gene or complement thereof of the nucleic acid.It is understood that other equivalent nucleic acids include those whichencode polypeptides having functions analogous to those described in theinstant invention using illustrative examples. Nucleic acids at least90%, more preferably 95%, and most preferably at least about 98-99%identical with a nucleic sequence represented in one of these sequencesor complement thereof are of course also within the scope of theinvention.

[0420] The invention also pertains to isolated nucleic acids comprisinga nucleotide sequence encoding certain polypeptides, variants and/orequivalents of such nucleic acids. The term equivalent is understood toinclude nucleotide sequences encoding functionally equivalentpolypeptides or functionally equivalent peptides having an activity of aprotein such as described herein.

[0421] Equivalent nucleotide sequences will include sequences thatdiffer by one or more nucleotide substitution, addition or deletion,such as allelic variants; and will, therefore, include sequences thatdiffer from the nucleotide sequence of the invention due to thedegeneracy of the genetic code.

[0422] Regardless of species, particularly preferred nucleic acids ofthe invention encode polypeptides that are at least 60%, 65%, 70%, 72%,74%, 76%, 78%, 80%, 90%, or 95% similar or identical to an amino acidsequence of the invention. For example, such nucleic acids can compriseabout 50, 60, 70, 80, 90, or 100 base pairs. Also within the scope ofthe invention, are nucleic acid molecules for use as probes/primer orantisense molecules (i.e. noncoding nucleic acid molecules), which cancomprise at least about 6, 12, 20, 30, 50, 60, 70, 80, 90 or 100 basepairs in length.

[0423] Another aspect of the invention provides a nucleic acid whichhybridizes under stringent conditions to a nucleic acid of theinvention. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6 or inMolecular Cloning: A Laboratory Manual, Cold Spring Harbor press (1989).For example, the salt concentration in the wash step can be selectedfrom a low stringency of about 2.0×SSC at 50° C. to a high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be increased from low stringency conditions at room temperature, atabout 22° C., to high stringency conditions at about 65° C. Bothtemperature and salt may be varied, or temperature and saltconcentration may be held constant while the other variable is changed.

[0424] Nucleic acids having a sequence that differs from the nucleotidesequences provided by the invention, or complement thereof due todegeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent peptidesbut differ in sequence from the sequence shown in the sequence listingdue to degeneracy in the genetic code. For example, a number of aminoacids are designated by more than one triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC each encodehistidine) may result in “silent” mutations which do not affect theamino acid sequence of an htrb polypeptide. However, it is expected thatDNA sequence polymorphisms that do lead to changes in the amino acidsequences of the subject polypeptides will exist among mammals. Oneskilled in the art will appreciate that these variations in one or morenucleotides (e.g., up to about 3-5% of the nucleotides) of the nucleicacids encoding polypeptides may exist among individuals of a givenspecies due to natural allelic variation.

[0425] 7.1 Probes and Primers

[0426] The nucleotide sequences determined from the cloning of genesfrom prokaryotic or eukaryotic organisms will further allow for thegeneration of probes and primers designed for use in identifying and/orcloning other homologs from other species. For instance, the presentinvention also provides a probe/primer comprising a substantiallypurified oligonucleotide, which oligonucleotide comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast approximately 12, preferably 25, more preferably 40, 50 or 75consecutive nucleotides of sense or anti-sense sequence of theinvention.

[0427] In preferred embodiments, the primers are designed so as tooptimize specificity and avoid secondary structures which affect theefficiency of priming. Optimized PCR primers of the present inventionare designed so that “upstream” and “downstream” primers haveapproximately equal melting temperatures such as can be estimated usingthe formulae: Tm (° C.)=81.5−16.6(log[Na⁺])+0.41(%G+C)−0.63(%formamide)−(600/length), for long polynucleotides; or Tm (°C.)=2(A+T)+4(G+C), for polynucleotides comprising less than 20 bases.Optimized primers may also be designed by using various programs, suchas “Primer3” provided by the Whitehead Institute for BiomedicalResearch.

[0428] 7.2. Vectors of the Invention

[0429] The invention further provides certain plasmids and vectors whichencode certain polypeptide products either in vitro or in vivo. The hostcell may be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of a mammalian pre-mRNA, encoding allor a selected portion of the full-length pre-mRNA, can be used toproduce a recombinant form of the pre-mRNA or other RNA sequence ofinterest via microbial or eukaryotic cellular processes. Ligating thepolynucleotide sequence into a gene construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial) cells,are standard procedures well known in the art.

[0430] Vectors that allow expression of a nucleic acid in a cell arereferred to as expression vectors. Typically, expression vectors usedfor expressing an RNA affinity substrate of the invention encode aribonucleoprotein assembly sequence and an affinity tag sequence whichcontains a nucleic acid encoding an RNA binding protein binding site,operably linked to at least one transcriptional regulatory sequence.Regulatory sequences are art-recognized. Transcriptional regulatorysequences are described in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0431] Suitable vectors for the expression of the RNA affinity substrateinclude plasmids of the types: pBR322-derived plasmids, pEMBL-derivedplasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derivedplasmids for expression in prokaryotic cells, such as E. coli.

[0432] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YBP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due to the presence of the pBR322 ori, and inS. cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markets such as ampicillin can beused.

[0433] The preferred expression vectors contain both prokaryoticpromoter sequences, such as a T7 promoter or an SP6 promoter so thatsynthetic RNA affinity substrates can be generated in vitro usingstandard methodologies. The various methods employed in the preparationof the plasmids and transformation of host organisms are well known inthe art. Fox other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. By Sambrook,Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989).

[0434] In some instances, it may be desirable to express a recombinantpolypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAeUVf1),and pBlueBac-derived vectors (such as the-β-gal containing pBlueBacIII).

[0435] When it is desirable to express only a portion of a protein, suchas a form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its ire vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing polypeptides in a host whichproduces MAP (e.g., E. coli ox CM89 or S. cerevisiae), or in vitro byuse of purified MAP (e.g., procedure of Miller et al., supra).

[0436] Moreover, the gene constructs of the present invention can alsobe used as part of a gene therapy protocol to deliver nucleic acidsencoding either an agonistic or antagonistic form of one of the subjectribonucloprotein complexes. Thus, another aspect of the inventionfeatures expression vectors for in vivo or in vitro transfection andexpression of a polypeptide in particular cell types so as toreconstitute the function of, or alternatively, abrogate the function ofa ribonucleoprotein complex in a tissue. Thus could be desirable, forexample, when the naturally-occurring form of the protein ismisexpressed or the natural protein is mutated and less active.

[0437] 8. Polypeptides of the Present Invention

[0438] The present invention provides methods to identify polypeptidesthat interact with a given ligand. Polypeptides identified through suchmethods can be produced in large quantity using any art-recognizedmethods, either as a purified polypeptide, or as a purified fusionpolypeptide with other polypeptides. All forms of polypeptides can beformulated, with an acceptable pharmaceutical excipient, into apharmaceutical composition using any art-recognized methods.

[0439] Such a purified polypeptide will be isolated from, or otherwisesubstantially free of other cellular proteins. The term “substantiallyfree of other cellular proteins” (also referred to herein as“contaminating proteins”) or “substantially pure or purifiedpreparations” are defined as encompassing preparations of polypeptideshaving less than about 20% (by dry weight) contaminating protein, andpreferably having less than about 5% contaminating protein. Functionalforms of the subject polypeptides can be prepared, for the first time,as purified preparations by using a cloned gene as described herein.

[0440] Preferred subject polypeptides have an amino acid sequence whichis at least about 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, or 95% identical or homologousto an amino acid sequence. Even more preferred subject polypeptidescomprise an amino acid sequence of at least 10, 20, 30, or 50 residueswhich is at least about 70, 80, 90, 95, 97, 98, or 99% homologous oridentical to an amino acid sequence. Such proteins can be recombinantproteins, and can be, e.g., produced in vitro from nucleic acidscomprising a nucleotide sequence identified by the methods of theinvention or homologs thereof. For example, recombinant polypeptidespreferred by the present invention can be encoded by a nucleic acid,which is at least 85% homologous and more preferably 90% homologous andmost preferably 95% homologous with a nucleotide sequence identified bythe methods of the invention- Polypeptides which are encoded by anucleic acid that is at least about 98-99% homologous with the sequenceidentified by the methods of the invention are also within the scope ofthe invention.

[0441] The scope of the invention also includes isoforms of the subjectpolypeptides encoded by splice variants. Such isoforms may haveidentical or different biological activities. Such isoforms may arise,for example, by alternative splicing of one or more gene transcripts.

[0442] Full length proteins or fragments corresponding to one or moreparticular motifs and/or domains or to arbitrary sizes, for example, atleast 5, 10, 20, 25, 50, 75 and 100, amino acids in length are withinthe scope of the present invention.

[0443] For example, isolated polypeptides can be encoded by all or aportion of a nucleic acid sequence. Isolated peptidyl portions ofproteins can be obtained by screening peptides recombinantly producedfrom the corresponding fragment of the nucleic acid encoding suchpeptides. In addition, fragments can be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-Moc or t-Boc chemistry. For example, a subject polypeptide may bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length. The fragments can be produced (recombinantly or bychemical synthesis) and tested to identify those peptidyl fragmentswhich can function as either agonists or antagonists of a wild-type(e.g., “authentic”) protein.

[0444] A polypeptide can be a membrane bound form or a soluble form. Apreferred soluble polypeptide is a polypeptide which does not contain ahydrophobic signal sequence domain. Such proteins can be created bygenetic engineering by methods known in the art. The solubility of arecombinant polypeptide may be increased by deletion of hydrophobicdomains, such as predicted transmembrane domains, of the wild typeprotein.

[0445] In general, polypeptides referred to herein as having an activity(e.g., are “bioactive”) of a protein are defined as polypeptides whichinclude an amino acid sequence encoded by all or a portion of thenucleic acid sequences and which mimic or antagonize all or a portion ofthe biological/biochemical activities of a naturally occurring protein.Examples of such biological activity include a region of conservedstructure referred to as the conserved domain.

[0446] Other biological activities of the subject proteins will bereasonably apparent to those skilled in the art. According to thepresent invention, a polypeptide has biological activity if it is aspecific agonist or antagonist of a naturally-occurring form of anprotein.

[0447] In addition to utilizing fusion proteins to enhanceimmunogenicity, it is widely appreciated that fusion proteins can alsofacilitate the expression of proteins, and accordingly, can be used inthe expression of the polypeptides of the present invention. Forexample, polypeptides can be generated as glutathione-S-transferase(GST-fusion) proteins. Such GST-fusion proteins can enable easypurification of the polypeptide, as for example by the rise ofglutathione-derivatized matrices (see, for example, Current Protocols inMolecular Biology, eds, Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).Additionally, fusion of polypeptides to small epitope tags, such as theFLAG or hemagluttinin tag sequences, can be used to simplifyimmunological purification of the resulting recombinant polypeptide orto facilitate immunological detection in a cell or tissue sample. Fusionto the green fluorescent protein, and recombinant versions thereof whichare known in the art and available commercially, may further be used tolocalize polypeptides within living cells and tissue.

[0448] The subject polypeptides may be produced by any method known inthe art. For example, a host cell transfected with a nucleic acid vectordirecting expression of a nucleotide sequence encoding the subjectpolypeptides can be cultured under appropriate conditions to allowexpression of the peptide to occur. Suitable media for cell culture arewell known in the art. The recombinant polypeptide can be isolated fromcell culture medium, host cells, or both using techniques known in theart for purifying proteins including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In, a preferred embodiment, the recombinant polypeptide is a fusionprotein containing a domain which facilitates its purification, such asGST fusion protein.

[0449] Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject polypeptides which function in a limited capacity as one ofeither an agonist (mimetic) or an antagonist in order to promote orinhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of proteins.

[0450] Homologs of each of the subject proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an receptor.

[0451] The recombinant polypeptides of the present invention alsoinclude homologs of the wild-type proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

[0452] Polypeptides may also be chemically modified to createderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of proteins can be prepared bylinking the chemical moieties to functional groups on amino acidside-chains of the protein or at the N-terminus or at the C-terminus ofthe polypeptide.

[0453] Modification of the structure of the subject polypeptides can befor such purposes as enhancing therapeutic or prophylactic efficacy,stability (e-g., ex vivo shelf life and resistance to proteolyticdegradation), or post-translational modifications (e.g., to alterphosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the polypeptides described in moredetail herein. Such modified peptides can be produced, for instance, byamino acid substitution, deletion, or addition. The substitutionalvariant may be a substituted conserved amino acid or a substitutednon-conserved amino acid.

[0454] For example, it is reasonable to expect that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. isosteric and/orisoelectric mutations) will not have a major effect on the biologicalactivity of the resulting molecule. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. In similarfashion, the amino acid repertoire can be grouped as (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine, (3)aliphatic=glycine, alanine, valine, leucine, isoleucine, serine,threonine, with serine and threonine optionally be grouped separately asaliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan;(5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine andmethionine. (see, for example, Biochemistry, 2^(nd) ed., Ed by L.Stryer, WFT Freeman and Co.: 1981). Whether a change in the amino acidsequence of a peptide results in a functional homolog (e.g., functionalin the sense that the resulting polypeptide mimics or antagonizes thewild-type form) can be readily determined by assessing the ability ofthe variant peptide to produce a response in cells in a fashion similarto the wild-type protein, or competitively inhibit such a response.Polypeptides in which more than one replacement has taken place canreadily be tested in the same manner.

[0455] This invention further contemplates the generation of sets ofcombinatorial mutants of the subject polypeptides as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g., homologs). The purpose of screening such combinatoriallibraries is to generate, for example, novel homologs which can act aseither agonists or antagonist, or alternatively, possess novelactivities all together. Thus, combinatorially-derived homologs can begenerated to have an increased potency relative to a naturally occurringform of the protein.

[0456] In one embodiment, the variegated library of variants isgenerated by combinatorial mutagenesis at the nucleic acid level, and isencoded by a variegated gene library. For instance, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofsequences therein.

[0457] There are many ways by which such libraries of potential homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into allappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential sequences. The synthesis of degenerate oligonucleotidesis well known in the art (see for example, Narang, SA (1983) Tetrahedron39:3; Itakura et al. (1981) Recombinant DNA, Proc 3d Cleveland Sympos.Macromolecules, ed: AG Walton, Amsterdam: Elsevier pp 273-289; Itakuraet al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198 :1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniqueshave been employed in the directed evolution of other proteins (see, forexample, Scott et al. (1990) Science 249 :386-390; Roberts et al. (1992)PNAS 89 :2429-2433; Devlin et al. (1990) Science 249 : 404-406; Cwirlaet al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,5,198,346, and 5,096,815).

[0458] Likewise, a library of coding sequence fragments can be providedfor any clone in order to generate a variegated population of fragmentsfor screening and subsequent selection of bioactive fragments. A varietyof techniques are known in the art for generating such 1 ibrary,including chemical synthesis. In one embodiment, a library of codingsequence fragments can be generated by (i) treating a double strandedPCR fragment of an coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule; (ii) denaturing thedouble stranded DNA; (iii) renaturing the DNA to form double strandedDNA which can include sense/antisense pairs from different nickedproducts; (iv) removing single stranded portions from reformed duplexesby treatment with S1 nuclease; and (v) ligating the resulting fragmentlibrary into an expression vector. By this exemplary method, anexpression library can be derived which codes for N-terminal, C-terminaland internal fragments of various sizes.

[0459] The invention also provides for reduction of the proteins togenerate mimetics, e.g., peptide or non-peptide agents, such as smallmolecules, which are able to disrupt binding of a subject polypeptidewith a molecule, e.g. target peptide. Thus, such mutagenic techniques asdescribed above are also useful to map the determinants of the proteinswhich participate in protein-protein interactions involved in, forexample, binding of the subject polypeptide to a target peptide. Toillustrate, the critical residues of a subject polypeptide which areinvolved in molecular recognition of its receptor can be determined andused to generate derived peptidomimetics or small molecules whichcompetitively inhibit binding of the authentic protein with that moiety.By employing, for example, scanning mutagenesis to map the amino acidresidues of the subject proteins which are involved in binding otherproteins, peptidomimetic compounds can be generated which mimic thoseresidues of the protein which facilitate the interaction. Such mimeticsmay then be used to interfere with the normal function of an protein.For instance, non-hydrolyzable peptide analogs of such residues can begenerated using benzodiazepine (e.g., see Freidinger et al. In Peptides:Chemistry and Biology, G.R-Marshall ed., ESCOM- Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher. Leiden,Netherlands, 1988), substituted gamma lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methyleue pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the American Peptide Symposium) PierceChemical Co, Rockland, Ill., 1985), b-tum dipeptide cores (Nagai et al.(1985) tetrahedron Lett 26:647; and Sato et al. (1986) 3 Chem Soc PerkinTrans 1:1231), and b-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Com:munl26:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71).

[0460] 9. Kits

[0461] The invention further provides kits for creating hybrid ligandswhich include a user-specified chemical ligand. The compound or agentcan be packaged in a suitable container. The kit can further compriseinstructions for using the kit to isolate binding proteins for theuser-specified ligand of the hybrid ligand.

[0462] One aspect of the invention provides a kit comprising apolynucleotide encoding at least one ligand binding domain and afunctional domain heterologous to the ligand binding domain which byitself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain, further comprising instructions 1) to synthesize ahybrid ligand of general structure R1-Y-R2, and 2) to test the bindingbetween the hybrid ligand and the ligand binding domain, wherein one ofR1 and R2 binds to or inhibits a kinase.

[0463] Another aspect of the invention provides a kit comprising apolynucleotide encoding at least one ligand binding domain and afunctional domain heterologous to the ligand binding domain which byitself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain, further comprising instructions 1) to synthesize ahybrid ligand of general structure R1-Y-R2, and 2) to test the bindingbetween the hybrid ligand and the ligand binding domain, wherein Y is ofthe general structure (CH₂—X—CH₂)_(n), where X represents O, S, SO, orSO₂, and n is an integer from 2 to 25.

[0464] Another aspect of the invention provides a kit comprising apolynucleotide encoding at least one ligand binding domain and afunctional domain heterologous to the ligand binding domain which byitself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain, further comprising instructions 1) to synthesize ahybrid ligand of general structure R1-Y-R2, and 2) to test the bindingbetween the hybrid ligand and the ligand binding domain, wherein thefunctional domain is Cub or Nux.

[0465] Another aspect of the invention provides a kit comprising: 1) acompound of general structure R1-Y-L, wherein Y is of the generalstructure (CH₂—X—CH₂)_(n) and L is a chemical group that is easilysubstituted by a different chemical group, and 2) instructions to usethe compound for the synthesis of a hybrid ligand R1-Y-R2 where R1 isdifferent from R2, and at least one of R1 and R2 is not a peptide.

[0466] 10. Business Methods

[0467] Other aspects of the invention provides for certain methods ofdoing business. In particular, practicing the methods of the inventionmay identify certain hybrid ligands, inhibitors and polypeptides. Thistechnical step, when combined with one of more additional steps providesfor novel approaches to conduct a pharmaceutical, agrochemical,biotechnological or preferable a life-science business. For example,such compositions identified by the method of the invention may betested for efficacy as therapeutics in a variety of disease models, thepotential therapeutic compositions then tested for toxicity and othersafety-profiling before formulating, packaging and subsequentlymarketing the resulting formulation for the treatment of disease.Alternatively, the rights to develop and market such formulations or toconduct such steps may be licensed to a third party for consideration.In certain other aspects of the invention, the hybrid ligands,inhibitors and polypeptides thus identified may have utility in the formof information that can be provided to a third party for considerationsuch that an improved understanding of the function or side effects ofsaid hybrid ligands, inhibitors and polypeptides in a biological ortherapeutic context.

[0468] By way of example, a particular preferably method of doingbusiness comprises:

[0469] (i) the identification of polypeptides binding to a hybrid ligandof general formula R1-Y-R2, wherein Y is of the general structure(CH₂—X—CH₂)_(n), R1 is different from R2, and at least one of R1 and R2is not a peptide, X═O, S, SO or SO₂, and wherein said polypeptides werepreviously not known to bind to such hybrid ligand, and

[0470] (ii) providing access to data, nucleic acids or polypeptidesobtained from such identification to another party for consideration.

EXAMPLES

[0471] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Oneskilled in the art, having read the specification and examples herein,will readily appreciate the possibility of numerous modifications,substitutions, combinations, permutations and improvements to themethods and compositions of the invention as herein disclosed. Suchmodifications, substitutions, combinations, permutations andimprovements are considered to be part of the present invention. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication) are hereby expressly incorporated by reference.

[0472] It will be clear to any skilled person that the biologicalsystems described herein are complex. The adaptation of the methodsexpressly exemplified herein below to similar systems as described abovemay involve a reasonable amount of experimentation, e.g. the adaptationof cell lines, construction of vectors, titration of concentrations,chemical modification of molecular species etc. In light of the complexnature of the systems contemplated herein, such experimentation cannotbe called undue, but will form a regular part of the establishment ofsuch methods in any laboratory. The skilled person will find sufficientguidance herein, and in the references cited as well as in standardtextbooks, to carry out modifications as necessary.

[0473] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of chemistry, cell biology,cell culture, molecular biology, microbiology and recombinant DNA, whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning: A LaboratoryManual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N.Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al.; U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S. J. Higgins eds. 1984); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer andWalker, eds., Academic Press, London, 1987).

[0474] The split ubiquitin technique was used to detect proteininteractions in vivo or in vitro. It is generally useful for all kindsof protein-protein interactions, but is particularly useful in caseswhen conventional yeast two-hybrid assay is problematic, i.e. membraneand cytosolic proteins, transcriptional activators or repressors, etc.

Example 1 Compound Synthesis

[0475] The following is a description of the synthesis of the hybridligands used herein. However, this description is to be understood asexemplary in nature, and shall in no way limit the scope of thecompounds according to the immediate invention. The person skilled inthe art will be readily able to envisage other synthetic routes tocompounds as provided by the present invention. For example, withoutlimitation, the building blocks H₂N—CH₂—(CH₂—O—CH₂—O—)_(n), —CH₂—N₃ withn=3, 6 and 12 are available from commercial sources (Toronto ResearchChemicals Inc., Toronto, Calif.; Fluka, Buchs, CH) and can be employedfor the synthesis of compounds of the general structure R1-Y-R2 withY=(—CH₂—O—CH₂)_(n), for example, without limitation, by a synthesisstrategy as used below in the synthesis of GPC 285937 following Scheme 2(See FIG. 1B).

[0476] In the compounds used herein, a methotrexate-moiety is linkedover 2 or more polyethylenglycol moieties as a linker to dexamethasone(GPC 285937), or to compounds known to bind to or inhibit CDKs. Thesepotential or known CDK inhibitors (CDKi) may be linked to methotrexatevia a linker in an orientation that preserves their activity towardsinhibition of CDKs (GPC 285985, IC₅₀ for CDK2 is approx. 180 nM), or inan orientation which abolishes this activity (GPC 285993, IC₅₀>10 μM).For comparison to previous results using methotrexate linked to othercompounds in a three hybrid assay (Lin et al., J. Am. Chem. Soc. 2000,122:4247-8), a hybrid ligand of methotrexate-linker-dexamethasone thatuses a metadibenzothioester as linker (Mtx-mdbt-Dex) was employed. Forthe establishment of the effect of varying exclusively the linker, twohybrid ligands were synthesized wherein methotrexate is linked to acompound with CDK inhibiting activity via a linker containing 3 (GPC286004) or 5 (GPC 286026) polyethylenglycol units.

[0477] Except where explicitly stated, all chemical reactants andsolvents used are available commercially from vendors the skilledartisan is well familiar with, for example Sigma-Aldrich (St. Louis,Mo., USA) and its subsidiaries.

[0478] Synthesis of GPC 285937 following Scheme I (See FIG. 1A)

[0479] Synthesis of tert-butyl(2R)-4-[N-(2-(2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-2-[(fluoren-9-ylmethoxy)carbonylamino]butanoate(3).

[0480] Fmoc-Glutamic acid a-tert-butyl ester (2.15 g, 5.1 mmol) wasdissolved in 10 ml dimethyl formamide (DMF) and1-amino-11-azido-3,6,9-trioxaundecane (1.0 g, 4.6 mmol) was added in 10ml DMF. To this solutionO-Benzotriazole-N,N,N′N′-tetramethyl-uronium-hexafluorophosphate (HBTU)(2.3 g, 6 mmol) and diisoproylethylamine (DIEA) (1.75 ml, 10 mmol) wereadded and the reaction stirred at room temperature for 2 hours. Thereaction mixture was diluted with 100 ml ethyl acetate and the organiclayer was washed with saturated sodium bicarbonate, 10% citric acid, andbrine, and then dried over magnesium sulfate and concentrated to a brownoil. The crude product (compound 3) was purified by flash silicachromatography (2% MeOH in EtOAc) to yield a light brown oil, 2.3 g, 3.7mmol, 80%.

[0481] Synthesis of tert-butyl(2R)-2-amino-4-[N-(2-t2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]butanoate (4).

[0482] Compound 3 (2.7 g, 4.3 mmol) was dissolved in 30 ml methylenechloride and 30 ml diethylamine was added. The reaction mixture wasstirred at room temperature for 2 h, and then concentrated to an oilunder reduced pressure. The residue was dissolved with diethyl ether andethyl acetate (ca. 50 ml ea.) and extracted with 10% citric acid. Theaqueous layer was neutralized to pH13 with 10N NaOH and extracted withethyl acetate. The organic layer was washed with brine, dried overmagnesium sulfate and concentrated under reduced pressure to give 1.6 gof a brown oil, 4.0 mmol, 92% (compound 4).

[0483] Synthesis of tert-butyl(2R)-4-[N-(2-{(2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]butanoate(6)

[0484] Compound 4 (140 mg, 0.35 mmol) and pteroic acid (compound 5) weredissolved together in 5 ml DMF andbenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBop) (0.26 g, 0.50 mmol) was added as a solid followed by DIEA (0.3ml, 1.7 mmol). The reaction mixture was stirred at room temperatureovernight, diluted with 30 ml ethyl acetate and the organic layer waswashed with 1N NaOH, brine, and then dried over magnesium sulfate andconcentrated under reduced pressure to give a brown oil. The crudeproduct was purified by reverse-phase (C8) HPLC to give 0.155 g of ayellow oil, approximately 70% pure (compound 6). The yield was 0.15mmol, 43%.

[0485] Synthesis of tert-butyl(2R)-4-[N-(2-{(2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]butanoate(7)

[0486] Compound 6 (0.155 g 70% pure, 0.15 mmol) was dissolved in 3 ml oftetrahydrofuran and 200 ml of water was added followed bytriphenylphosphine (130 mg, 0.5 mmol). The reaction mixture was stirredat room temperature for 16 hours, diluted with 20 ml diethyl ether andthe organic layer extracted with 10% citric acid. Aqueous layer wasneutralized to pH 12 with ION NaOH and extracted with ethyl acetate. Theorganic layer was washed with brine, dried over magnesium sulfate andconcentrated under reduced pressure to yield an oil. The crude productwas purified by reverse-phase (C8) HPLC to give 16 mg of a yellow oil,0.022 mmol, 15% (compound 7).

[0487] Synthesis of4-((2,4-diamino-6-pteridinylmethyl)methylamino)benzoyl-L-Gln(11-(9-fluoro-11b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxamido)-3,6,9-trioxoundecyl)(9, GPC 285937)

[0488] 9-fluoro-11 b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxylic acid(compound 8) 12 mg, 0.032 mmol) and compound 7 (15 mg, 0.021 mmol) werecombined in 0.5 ml DMF and PyBop (20 mg, 0.038 mmol) was added followedby 0.017 ml DIEA (0.1 mmol). The reaction mixture was stirred at roomtemperature for 16 hours and then diluted with 10 ml ethyl acetate. Theorganic layer was washed with 0.2 N NaOH and brine, and thenconcentrated under reduced pressure to give an oil. This oil wasdissolved in 2 ml 1:1 TFA:CH₂Cl₂ and let stand for 1 hour. The solventwas removed under reduced pressure and the residue was purified byreverse-phase (C8) HPLC to give 2.8 mg of product, 0.0028 mmol, 13%(compound 9).

[0489] Synthesis of GPC 285937 following Scheme 2 (See FIG 1B)

[0490] Synthesis of tert-butyl(2S)-4-[N-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-2-({4-[N-methyl(phenylmethoxy)carbonylamino]phenyl}carbonylamino)butanoate(11)

[0491] Compound 4 (0.81 g, 2.0 mmol) and4-carboxybenzylmethylaminobenzoic acid (compound 10) (0.61 g, 2.1 mmol)were dissolved in 10 ml DMF. To this solution, HBTU (1.0 g, 2.6 mmol)was added as a solid followed by DIEA (0.8 ml, 4.6 mmol). The reactionmixture was stirred overnight at room temperature, diluted with ethylacetate and the organic layer was washed with 0.5N NaOH, brine, driedover magnesium sulfate and concentrated under reduced pressure to give abrown oil. The crude product was purified by flash silica chromatography(5% MeOH in EtOAc) to yield a brown oil (1.03 g, 1.5 mmol, 77%, compound11).

[0492] Synthesis of tert-butyl(2S)-4-[N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-2-({4-[N-methyl(phenylmethoxy)carbonylamino]phenyl}carbonylamino)butanoate(12)

[0493] Compound 11 (1.0 g, 1.49 mmol) was dissolved in 50 ml MeOH and130 mg 10% Pd/C added. The reaction mixture was shaken under 40 psihydrogen for 16 hours, the catalyst was filtered off, and the filtratewas concentrated under reduced pressure to give 0.75 g (1.47 mmol, 98%)of a colorless oil (compound 12).

[0494] Synthesis of 4-methylaminobenzoyl-L-Gln(11-(9-fluoro-11b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxamido)-3,6,9-trioxoundecyl)tert-butyl ester (13)

[0495] Compound 12 (0.75 g, 1.47 mmol) was dissolved in DMF with9-fluoro-11b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxylic acid (8)(0.60 g, 1.6 mmol) and to this solution HBTU was added (0.75 g, 2 mmol)followed by DIEA (0.35 ml, 2 mmol). The reaction mixture was stirredovernight at room temperature, diluted with ethyl acetate, and theorganic layer was washed with saturated sodium bicarbonate, brine, andconcentrated under reduced pressure to give an orange oil. The crudeproduct was purified by flash silica chromatography (10% MeOH in EtOAc)to yield 0.54 g of a white foam (0.62 mmol, 42%, compound 13).

[0496] Synthesis of 2,4-diamino-6-(bromomethyl)pteridine hydrobromide(14)

[0497] Synthesis of 2,4-diamino-6-(bromomethyl)pteridine hydrobromide(compound 14) was carried out in two steps individually described in theliterature (Taghavi and Pfleiderer, Tetrahedron Lett., 1997, 38:6835-36;Taylor and Portnoy, J. Org. Chem., 1973, 38:806).

[0498] Synthesis of4-((2,4-diamino-6-pteridinylmethyl)methylamino)benzoyl-L-Gln(11-(9-fluoro-11b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxamido)-3,6,9-trioxoundecyl) tert-butyl ester (15)

[0499] Compound 13 (0.54 g, 0.62 mmol) and 0.41 g compound 14 (1.2 mmol)were combined in 8 ml dimethylacetamide and heated to 60° C. for 6hours. Diethyl ether (100 ml) was added and a precipitate formed. Thesupernatant was decanted off and the residue was purified by silicachromatography (1:10:89, saturated NH₄OH:MeOH:CH₂Cl₂) to yield 0.35 g ofa yellow solid (0.33 mmol, 54%, compound 15).

[0500] Synthesis of4-((2,4-diamino-6-pteridinylmethyl)methylamino)benzoyl-L-Gln(11-(9-fluoro-11b,17-dihydroxy-16a-methyl-3-oxoandrosta-1,4-diene-17b-carboxamido)-3,6,9-trioxoundecyl) (9, GPC 285937)

[0501] Compound 15 (0.35 g, 0.33 mmol) was dissolved in 20 ml (1:1:8:10,H₂O:Me₂S:CH₂Cl₂:TFA) and the reaction was stirred for 1 hour at roomtemperature. The solvent was removed under reduced pressure and theresidue was dissolved in MeOH and purified by reverse-phase (C8) HPLC.The fractions containing product were concentrated to a minimal volumeand then lyophilized to give 0.30 g of a yellow solid (0.27 mmol, 83%).

[0502] Synthesis of GPC 285985 following Scheme 3 (See FIG. 1C)

[0503] Synthesis of Ethyl2-methyl-2-(4-{[3-(methylethyl)-4-oxo-1-(2,4,6-trichlorophenyl)(5-hydropyrazolo[5,4-d]pyrimidin-6-yl)]methyl}phenoxy) propanoate (17)

[0504] Compound 16 (2.5 g, 7.2 mmol) and ethyl2-{4-[(ethoxycarbonyl)methyl]phenoxy}-2-methylpropanoate (4.5 g, 15.3mmol) were dissolved in 15 ml of ethanol and 5.8 ml of a 2.66M solutionof sodium ethoxide in ethanol (15.3 mmol) was added. The reactionmixture was heated to reflux for 5 hours, cooled to room temperature andlet stand overnight. The reaction mixture was then diluted with ethylacetate and washed with water and brine, dried over magnesium sulfate,filtered and concentrated to 1.6 g (2.8 mmol, 38%) of a beige solid(compound 17).

[0505] Synthesis of 2-methyl-2-(4-{[3-(methylethyl)-4-oxo-1-(2,4,6-trichlorophenyl)(5-hydropyrazolo[5,4-d]pyrimidin-6-yl)]methyl}phenoxy)propanoic acid(18)

[0506] Compound 16 (1.6 g, 2.8 mmol) was dissolved in 30 ml dioxane, 10ml methanol and treated with 5 ml (5 mmol) of 1N NaOH. The reaction wasstirred at room temperature overnight, then diluted with ethyl acetateand washed with 1N HCl and then brine. The organic layer was dried overmagnesium sulfate, filtered and concentrated to a solid (1.4 g, 2.5mmol, 91%, compound 18).

[0507] Synthesis of Tert-Butyl(2R)-2-{[4-(methylamino)phenyl]carbonylamino}-4-(N-{2-[2-(2-{2-[2-methyl-2-(4-{([3-(methylethyl)-4-oxo-1-(2,4,6-trichlorophenyl)(5-hydropyrazolo[5,4-d]pyrimidin-6-yl)]methyl}phenoxy)propanoylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)butanoate(19)

[0508] Compound 18 (0.70 g, 1.3 mmol) and compound 12 (0.63 g, 1.2 mmol)were dissolved in dimethyl formamide and HBTU (0.75 g, 2 mmol) was addedfollowed by diisopropylethylamine (0.5 ml, 2.9 mmol). The reactionmixture was stirred at room temperature for 3 days, diluted with ethylacetate and then washed with 0.5N NaOH and brine. The organic layer wasdried over magnesium sulfate, filtered and concentrated to an oil whichwas purified by flash silica chromatography (5 to 10% MeOH/EtOAc) togive 430 mg (0.41 mmol, 34%) of brown foam (compound 19).

[0509] Synthesis of(2R)-2-[4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]-4-(N-{2-[2-{2-2-[2-methyl-2-(4-{[3-(methylethyl)-4-oxo-1-(2,4,6-trichlorophenyl)(5-hydropyrazolo[5,4-d]pyrimidin-6-yl)]methyl}phenoxy)propanoylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)butanoicAcid (20, GPC 285985)

[0510] Compound 19 (0.43 g, 0.41 mmol) was dissolved in 10 ml dimethylacetamide and 0,27 g compound 14 (0.80 mmol) was added to the reactionmixture as a solid. The reaction mixture was heated to 60° C. for 5hours, then let cool to room temperature and 100 ml diethyl ether added.The supernantant was decanted off leaving a dark brown residue which wastaken up in 10 ml of a cleavage cocktail (10:10:1:1 TFA:CH₂Cl₂: Me₂S:H₂O) and stirred for one hour. Solvent removed under reduced pressure,and the residue was purified by RPHPLC. Fractions containing the productwere combined, concentrated to a small volume and lyophilized to yield ayellow solid (101 mg, 0.086 mmol, 21%, compound 20).

[0511] Synthesis of GPC 286004 and GPC 286026 following Schemes 4 and 5(See FIGS. 1D and 1E)

[0512] Synthesis of Ethyl2-{4-[(4-nitro-1,3-dioxo-2-hydrocyclopenta[3,4-a]benzen-2-yl)carbonyl]phenoxy}acetate(21)

[0513] Ethyl 2-[4-(4,4,4-trifluoro-3-oxobutanoyl)phenoxy]acetate (31.9g, 0.1 mol) was combined with 19.3 g (0.1 mol) 3-nitrophthallicanhydride and 57 ml (0.6 mol) of acetic anhydride added. The slushysuspension was stirred at 0° C. and 28 ml (0.2 mol) triethyl amineadded. The reaction mixture became homogenous and red and was stirred atroom temperature overnight at which time 600 ml 1N HCl added. Theresulting tacky suspension was stirred for 2 hours and the precipitatebecame a granular solid which was filtered off, resuspended in 200 mlethanol, heated to reflux and then cooled to 0° C. A yellow solid wasfiltered off, washed with ethanol (3×40 ml) and dried to 12.7 g, 32mmol, 32% yield (compound 21).

[0514] Synthesis of Ethyl2-{4-[(4-amino-1,3-dioxo-2-hydrocyclopenta[3,4-a]benzen-2-yl)carbonyl]phenoxy}acetate(22)

[0515] Compound 21 (12.7 g, 32 mmol) was partially dissolved in 600 mlethyl acetate and 1.5 g of 10% Pd/C added. The reaction was stirredunder a balloon of H₂ overnight. The balloon was recharged with H₂ andstirred for 24 hours more. The reaction was filtered through celite withthe help of THF and CH₂Cl₂ to dissolve the product, and the filtrate wasconcentrated to 10.7 g (29.1 mmol, 91%) of solid (compound 22).

[0516] Synthesis of ethyl2-[4-({4-[(morpholin-4-ylamino)carbonylamino]-],3-dioxo-2-hydrocyclopenta[3,4-a]benzen-2-yl}carbonyl)phenoxy]acetate(23)

[0517] Compound 22 (6.4 g, 17.4 mmol) was combined in acetonitrile with4-nitrophenyl morpholine4-carboxylate (containing 1 eq. triethylammonium chloride impurity) (8.0 g, 19.8 mmol) and dimethylaminopyridine(0.20 g, 1.6 mmol) was added. The suspension was heated to reflux for 3hours, cooled to 0° C. and a yellow solid filtered off. This solid waswashed with a minimum of cold acetonitrile, and dried to 6.7 g, 13.5mmol, 78% (compound 23).

[0518] Synthesis of2-[4-({4-[(morpholin-4-ylamino)carbonylamino]-1,3-dioxo-2-hydrocyclopenta[3,4-a]benzen-2-yl)carbonyl)phenoxy]aceticAcid (24)

[0519] Compound 23 (6.7 g, 13.5 mmol) was dissolved in 200 ml dioxaneand 20 ml (20 mmol) 1N NaOH added. The reaction mixture was stirred forone hour. The white suspension was diluted with 1 l ethyl acetate andwashed with 1N HCl and brine. The organic layer was dried over magnesiumsulfate, filtered and concentrated to a yellow solid (6.3 g, 13.5 mmol,100%, compound 24).

[0520] Synthesis of2-(4-(5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)aceticAcid (25)

[0521] Compound 24 (6.5 g, 13.5 mmol) was dissolved in 200 ml THF, 100ml DMSO and treated with 4 g (80 mmol) hydrazine hydrate and 190 mg, (1mmol) p-toluenesulfonic acid hydrate. The reaction mixture was heated to60° C. for 5 hours, let cool to room temperature and 600 ml Et₂O added.The resulting suspension was then filtered, the precipitate washed with1N HCl and dried under vacuum to yield 4.0 g (8.6 mmol, 64%) of yellowsolid (compound 25).

[0522] Synthesis of tert-butyl(2S)-4-(N-{2-[2-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)-2-{[4-(methylamino)phenyl]carbonylamino}butanoate(26)

[0523] Compound 26 was synthesized by an analogous procedure as employedfor compound 12, but using1-amino-17-azido-3,6,9,12,15-pentaoxaheptadecane instead of1-amino-11-azido-3,6,9-trioxaundecane in the first step of synthesis.

[0524] Synthesis of tert-butyl(2S)-2-{[4-(methylamino)phenyl]carbonylamino}-4-(N-{2-[2-(2-{2-[2-(4-{5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethyl}carbamoyl)butanoate(27)

[0525] Compound 12 (0.71 g, 1.4 mmol) and compound 25 (0.57 g, 1.2 mmol)were dissolved in 10 ml DMF and HBTU (0.8 g, 2.1 mmol) was added as asolid followed by DIEA (0.52 ml, 3 mmol). The reaction mixture wasstirred at room temperature for 3 days, diluted with EtOAc and theorganic phase washed with saturated NaHCO₃. The aqueous layer was backextracted with EtOAc twice and the combined organic layers dried overMgSO₄, filtered and concentrated to an oil. This oil was purified byflash silica chromatography (2 to 5% MeOH/EtOAc) to give an orange oil(0.50 g, 0.52 mmol, 44%, compound 27).

[0526] Synthesis of tert-butyl(2S)-2-{[4-(methylamino)phenyl]carbonylamino}-4-{N-[2-(2-{2-[2-(2-{2-[2-(4-{5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoate(28)

[0527] Compound 25 (0.60 g, 1 mmol) and compound 26 (0.46 g, 1 mmol)were dissolved in 10 ml DMF and HBTU (0.7 g, 1.8 mmol) was added as asolid followed by DIEA (1.0 ml, 5.7 mmol). The reaction mixture wasstirred at room temperature overnight, diluted with EtOAc and theorganic phase washed with 0.5N NaOH, brine, dried over MgSO₄, filteredand concentrated to an oil. This oil was purified by flash silicachromatography (10 to 20% MeOH/EtOAc) to give a yellow foam (0.65 g,0.62 mmol, 62%, compound 28).

[0528] Synthesis of tert-butyl(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]-4-{N-[2-(2-{2-[2-(2-{4-[5-(methoxycarbonylamino)-4-oxoindeno[3,2-c]pyrazol-3-yl]phenoxy}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoate(29)

[0529] Compound 27 (0.50 g, 0.52 mmol) was dissolved indimethylacetamide and 0,33 g of compound 14 (1.0 mmol) was added to thereaction mixture as a solid. The reaction mixture was heated to 60° C.for 6 hours, then let cool to room temperature and 80 ml diethyl etheradded. The supernantant was decanted off leaving a dark brown residue,which was purified by flash silica chromatography (5 to 10% MeOH/CH₂Cl₂then 5 to 10% MeOH/CH₂Cl₂ w/1% NH₄OH) to give 0.33 g (0.29 mmol, 56%) ofa yellow solid (compound 29).

[0530] Synthesis of Tert-butyl(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methy]methylamino}phenyl)carbonylamino]-4-{N-[2-(2-{2-[2-(2-{2-[2-(4-{5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoate (30)

[0531] Compound 28 (0.65 g, 0.62 mmol) was dissolved indimethylacetamide and 0,4 g of compound 14 (1.2 mmol) was added to thereaction mixture as a solid. The reaction mixture was heated to 60° C.for 6 hours, then let cool to room temperature and 80 ml diethyl etheradded and let stand for 3 days. The supernantant was decanted offleaving a dark brown residue, which was purified by flash silicachromatography (5 to 10% MeOH/CH₂Cl₂ then 5 to 10% MeOH/CH₂Cl₂ w/1%NH₄OH) to give 0.45 g (0.37 mmol, 60%) of a yellow solid (compound 30).

[0532] Synthesis of(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonyl-amino]-4-{N-[2-(2-{2-[2-(2-{(4-[5-(methoxy-carbonyl-amino)-4-oxoindeno[3,2-c]pyrazol-3-yl]phenoxy}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoy}butanoicAcid (31, GPC 286004)

[0533] Compound 29 (0.33 g, 0.29 mmol) was treated with 20 ml of acleavage cocktail (10:10:1:1 TFA:CH₂Cl₂: Me₂S: H₂O). After one hour,solvent removed and the residue purified by RPHPLC. Fractions containingthe product were combined, concentrated to a small volume andlyophilized to yield a yellow solid (0.19 g, 0.18 mmol, 61%, compound31).

[0534] Synthesis of(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonyl-amino]-4-{N-[2-(2-{2-[2-(2-{2-[2-(4-{5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-3-yl}phenoxy)acetylamino]ethoxy}ethoxy)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoicAcid (32, GPC286026)

[0535] Compound 30 (0.45 g, 0.37 mmol) was treated with 20 ml of acleavage cocktail (10:10:1:1 TFA:CH₂Cl₂: Me₂S: H₂O). After one hour, thesolvent was removed and the residue purified by RPHPLC. Fractionscontaining the product were combined, concentrated to a small volume andlyophilized to yield a yellow solid (0.23 g, 0.18 mmol, 49%, compound32).

[0536] Synthesis of GPC 285993 following Scheme 6 (See FIG. 1F)

[0537] Synthesis of1-(4-Benzyloxy-phenyl)-4,4,4-trifluoro-butane-1,3-dione

[0538] 45.2 g 1-(4-Benzyloxy-phenyl) ethanone (200 mmol) was taken up inTHF (250 mL) and treated with CF₃CO₂Et (30 ml, 250 mmol). The solutionwas cooled to 0° C. and treated with 2.66 M NaOEt (94 ml, 250 mmol)solution over 1 h. The ice bath was removed and the solution was stirredat room temperature for 4 h. The reaction was poured into 1N HCl (1000ml) and extracted with EtOAc (1500 ml). The organic layer was washedwith brine, dried and evaporated to yield 64.2 g1-(4-Benzyloxy-phenyl)-4,4,4-trifluoro-butane-1,3-dione (200 mmol, 100%yield).

[0539] Synthesis of4-nitro-2-[(4-hydroxyphenyl)carbonyl]-2-hydrocyclopenta[1,2-a]benzene-1,3-dione(33)

[0540] 64 g 1-(4-Benzyloxy-phenyl)-4,4,4-trifluoro-butane-1,3-dione (200mmol) was suspended in Ac₂O (114 mL, 1.2 mol) and treated with3-nitropthalic anhydride (28.6 g, 200 mmol). The suspension was cooledto 0° C. and treated slowly with Et₃N (56 ml, 400 mmol). The reactionwas stirred at room temperature for 16 h, then poured into ice/3N HCl(500 ml) and stirred vigorously for 1 h. The precipitate was filteredand washed with water. The precipitate was suspended in boiling ethanol(450 ml) for 10 min, then cooled to 0° C. for 2 h and filtered. Thesolid was washed with cold ethanol and dried under vacuum to yield 34 g(72 mmol, 36% yield, compound 33).

[0541] Synthesis of4-amino-2-[(4-hydroxyphenyl)carbonyl]-2-hydrocyclopenta[1,2-a]benzene-1,3-dione(34)

[0542] Compound 33 (32.1 g, 67.6 mmol) was dissolved in 1500 ml EtOAcand 3.2 g 10% Pd/C added. The reaction mixture was stirred under anatmosphere (balloon) of H₂ for 3 days. Methanol was added to aiddissolution and the reaction mixture was filtered through celite. Thefiltrate was concentrated to 19 g (67 mmol, 100%) of an orange solid(compound 34).

[0543] Synthesis ofN-{2-[(4-hydroxyphenyl)carbonyl]-1,3-dioxo(2-hydrocyclopenta[2,1-b]benzen-4-yl)}(morpholin-4-ylamino)carboxamide(35)

[0544] Compound 34 (10.0 g, 35.3 mmol) was dissolved in acetonitrilewith 4-nitrophenyl morpholine4-carboxylate (containing 1 eq. triethylammonium chloride impurity) (13.0 g, 32.1 mmol) anddimethylaminopyridine (0.60 g, 5.4 mmol) was added. The reaction mixturewas heated to reflux for 3 hours, cooled to room temperature and a palegreen solid filtered off and dried to 7.5 g (18.3 mmol, 57%, compound35).

[0545] Synthesis ofN-[3-(4-hydroxyphenyl)-4-oxoindeno[3,2-c]pyrazol-5-yl](morpholin-4-ylamino)carboxamide(36)

[0546] Compound 35 (7.5 g, 18.3 mmol) was suspended in 200 ml THF andhydrazine hydrate (4.5 g, 90 mmol) was added followed byp-toluenesulfonic acid hydrate (340 mg, 1.8 mmol). The reaction mixturewas heated to reflux overnight (homogenous solution), let cool to roomtemperature and a precipitate formed, which was filtered off to give 1.2g of product. The filtrate was concentrated to a solid, suspended inEtOAc and filtered. This solid was purified by flash silicachromatography (5 to 10% MeOH/EtOAc) to give 2.2 g more of product. Thecombined yield was 3.3 g, 8.4 mmol, 46% (compound 36).

[0547] Synthesis of Ethyl2-{(3-(4-hydroxyphenyl)-5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-2-yl}acetate (37)

[0548] Compound 36 (2.2 g, 5.6 mmol) was dissolved in 50 ml acetone, 10ml THF, and 10 ml DMF and Cs₂CO₃ (1.8 g, 5.6 mmol) was added followed byethyl bromoacetate (0.93 g, 5.6 mmol). The reaction mixture was stirredfor 2 hours, diluted with ethyl acetate, and the organic layer washedwith 1N HCl, brine, dried over MgSO₄, filtered and concentrated to ayellow solid. The solid was purified by flash silica chromatography (2to 3 to 4% MeOH/CH₂Cl₂) to give 1.2 g (2.4 mmol, 44%) of a yellow solid(compound 37).

[0549] Synthesis of2-{3-(4-hydroxyphenyl)-5-[(morpholin-4-ylamino)carbonylamino]-4-oxoindeno[3,2-c]pyrazol-2-yl}aceticAcid (38)

[0550] Compound 37 (1.2 g, 2.4 mmol) was dissolved in 60 ml 3:2:1;dioxane:ethanol:DMSO and 12 ml 0.5 N NaOH added and the reaction becamered. The reaction mixture was stirred at room temperature for one hour,diluted with EtOAc and washed with 1N HCl. The aqueous layer was backextracted once with ethyl acetate and the combined organic layers driedover MgSO4 and concentrated to an orange solid. The solid was trituratedwith 10 ml MeOH/100 ml Et₂O, filtered off and dried to a solid (1.1 g,2.4 mmol, 100%, compound 38).

[0551] Synthesis of Tert-Butyl(2S)-4-{N-[2-(2-{2-[2-(2-{3-(4-hydroxyphenyl)-5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-2-yl}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-{[4-(methylamino)phenyl]carbonylamino}butanoate (39)

[0552] Compound 38 (0.52 g, 1.1 mmol) and compound 12 (0.55 g, 1.1 mmol)were dissolved in DMF and HBTU (0.8 g, 2.1 mmol) was added as a solidfollowed by DIEA (0.52 ml, 3 mmol). The reaction mixture was stirred atroom temperature overnight, diluted with EtOAc and the organic phasewashed with saturated NaHCO₃, brine, dried over MgSO₄, filtered andconcentrated to an oil. This oil was purified by flash silicachromatography (1 to 2 to 3 to 4 to 5% MeOH/CH₂Cl₂) to give a yellowfoam (0.45 g, 0.47 mmol, 43%, compound 39).

[0553] Synthesis of tert-butyl(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenylcarbonylamino]-4-{N-[2-(2-{2-[2-(2-{3-(4-hydroxyphenyl)-5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-2-yl}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoate(40)

[0554] Compound 39 (0.45 g, 047 mmol) was dissolved in 8 mldimethylacetamide and 0,2 g compound 14 (0.60 mmol) was added to thereaction mixture as a solid. The reaction mixture was heated to 60° C.for 6 hours, then let cool to room temperature and diethyl ether added.The supemantant was decanted off leaving a dark brown residue, which waspurified by flash silica chromatography (5 to 10% MeOH/CH₂Cl₂ then 5 to10% MeOH/CH₂Cl₂ w/1% NH₄OH) to give 0.32 g (0.27 mmol, 56%) of yellowsolid (compound 40).

[0555] Synthesis of(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl]methylamino}phenyl)carbonylamino]-4-{(N-[2-(2-{(2-[2-(2-{3-(4-hydroxyphenyl)-5-[(N-morpholin-4-ylcarbamoyl)amino]-4-oxoindeno[3,2-c]pyrazol-2-yl}acetylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamoyl}butanoic Acid (41, GPC 285993)

[0556] Compound 40 (0.30 g, 0.27 mmol) was treated with 20 ml of acleavage cocktail (10:10:1:1 TFA:CH₂Cl₂: Me₂S: H₂O). After one hour,solvent removed and the residue purified by RPHPLC. Fractions containingthe product were combined, concentrated to a small volume andlyophilized to yield a yellow solid (78 mg, 0.073 mmol, 27%, compound41).

[0557] Synthesis of Mtx-(CH₂—O—CH₂)₅-Purvalanol B (42) according toScheme 7 (See FIG. 1G)

[0558] Purvalanol B was synthesized following the methods described inChang et al. & Schultz (1999), Chem. Biol. 6:361-375. Purvalanol B (0.35g, 0.8 mmol) and(S)-4-{2-[2-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethylcarbamoyl}-2-(4-methylamino-benzoylamino)-butyricacid tert-butyl ester (0.54 g, 0.9 mmol) were mixed in 2 ml of dimethylacetamide (DMA) to which was added diisopropylethylamine (0.31 g, 2.4mmol) and HBTU (0.36 g, 1.0 mmol). The reaction mixture was stirredovernight, the resulting solution diluted with 8 ml ethyl acetate andwashed with saturated aqueous NaHCO₃ and brine. The organic layer wasseparated, dried with anhydrous magnesium sulfate, filtered andconcentrated. The crude product was purified via silica gelchromatography eluting with 5% MeOH/1% NH₄OH/94% CH₂Cl₂ to give 0.48 gof compound 43 (60% yield)

[0559] Compound 43 (0.48 g, 0.48 mmol) and6-Bromomethyl-pteridine-2,4-diamine hydrobromide (0.32 g, 0.96 mmol)were combined in DMA and heated to 60° C. for 4 hours. The reactionvessel was allowed to cool, the mixture diluted with diethyl ether andwashed with saturated aqueous NaHCO₃ and brine. Organic layer separated,dried with anhydrous magnesium sulfate, filtered and concentrated. Crudeproduct purified via silica gel chromatography eluting with 10% MeOH/2%NH₄OH/84% CH₂Cl₂ to give 0.29 g of compound 44 (50% yield.)

[0560] Compound 44 (0.29 g, 0.24 mmol) was placed in a mixture of 4.5 mltrifluoroacetic acid, 4.5 ml methylene chloride, 0.5 ml water and 0.5 mldimethylsulfide at room temperature and stirred for 30 minutes. Thesolution was then concentrated and the residue diluted with 20 ml of a1:1 acetonitrile/water mixture. The solution was purified viapreparative HPLC to give 69.3 mg of compound 42 after lyophilization.

Example 2 Measurement of Affinities of Hybrid Ligands for SelectedBinding Proteins

[0561] To demonstrate the characterization of affinity between hybridligands and proteins they bind to, we analyzed the binding of GPC 285985to its expected binding partners DHFR and CDK2/E (cyclin dependentkinase 2/cyclin E complex). The analysis was performed on a BIACORE 2000SPR-Biosensor (Biacore, Uppsala, Sweden) at 22° C. using a runningbuffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM DTT and 0.005%Tween20 (protein grade, Calbiochem). Vector pQE40 (Qiagen, Hilden,Germany), comprising the gene encoding DHFR fused to a his₆-tag, wastransformed into E. coli and the His₆-DHFR fusion protein purifiedfollowing manufacturers protocols. His₆-DHFR was subsequently coupled atpH 4.6 to the dextrane-surface of a CM5 sensor-chip (Biacore, Uppsala,Sweden; research grade) according to manufacturers instructions. Theloading density reached 1100 RU (Resonance Units). A 10 μM solution ofGPC 285985 was allowed to pass over the DHFR-loaded chip surface for 5minutes at a flow rate of 30 μl/min, followed by 5 minutes of runningbuffer at the same flow rate. A profile for adsorption and desorption ofGPC 285985 on DHFR was obtained and stored. Non-specific binding of GPC285985 was assessed using a CM5-surface with deactivated COOH-groups.The resulting sensorgram (not shown) demonstrated specific and highaffinity binding of the hybrid ligand to the DHFR-coated surface.

[0562] In order to characterize the binding of GPC 285985 to otherproteins, the CM5-DHFR surface was first loaded with GPC 285985 bypassing a 10 μM solution of GPC 285985 over the chip surface for 5minutes at a flow rate of 10 μl/min Then, CDK2/E complex, for examplepurified from baculovirus infected cells expressing CDK2 and Cyclin E(Sarcevic et al., J. Biol. Chem., 1997 272:33327-37), was diluted inrunning buffer to obtain eight distinct protein concentrations rangingfrom 6 nM to 750 nM, which were then each allowed to pass over thesensor surface consecutively for 5 min each, followed by 5 min ofrunning buffer at the same flow rate. The association and dissociationof the CDK2/E complex onto the CM5-DHFR::GPC 285985-loaded chip surfacewas measured at a flow rate of 301 μl/min After eachassociation/dissociation experiment, the chip was regenerated to removebound protein by two consecutive injections of 3 Mguanidinium-hydrochloride (20 sec, 30 μl/min) before the next sample wasloaded. Non-specific binding was assessed using a CM5-surface loadedwith DHFR only.

[0563] The data were analyzed using the Bioevaluation software version3.1 (Biacore AB, Uppsala, SE). The curves were normalized to theinjection start, and the non-specific binding to the DHFR-loaded controlsurface and the background line drift resulting from desorption of GPC285985 from the CM5-DHFR during the 10 min run were subtracted. Theassociation and dissociation rates were determined separately orglobally using a Langmuir 1:1 binding model as provided by theBioevaluation software 3.1. The affinities (KD) were calculated usingthe equation:

K _(D) =k _(diss) /k _(ass)

[0564] This association/disassociation experiment gave a K_(D) of 8.0 nMfor the binding of GPC 285985 to CDK2, confirming the high specificityof the hybrid ligand GPC 285985 for CDK2. FIG. 2 shows as an example theresults of an analogous association/dissociation experiments obtainedfor the binding of CDK4/D1 to the CM5-DHFR::GPC 285985-loaded chip. TheK_(D) for the binding of GPC 285985 to the CDK4/D1 complex wascalculated from these data as 920 nM. This confirms the expected resultsof strong binding of GPC 285985 to DHFR and CDK2, but weak binding tothe closely related kinase CDK4. The CDK4/CyclinD1 complex was purifiedfor example from baculovirus infected cells expressing (Konstantinidiset al., J. Biol. Chem., 1998, 273:26506-15).

Example 3 Pull Down of Polypeptides which Interact with a Hybrid Ligandof the Invention

[0565] To show the usefulness of the hybrid ligands of the invention forso called “pull-down” experiments, a method was devised where the hybridligand was incubated with strep-tagged DHFR and cell lysates, complexessubsequently pulled down using a Streptactin-coated resin, unboundproteins washed out and the bound proteins eluted by competitivedisplacement of the hybrid ligand. Therein, in terms of the presentinvention, DHFR and Methotrexate represents P1 and R1, respectively, theknown protein-ligand combination, the linker Y is represented by a—(CH₂—O—CH₂)₅-group, R2, the small molecule for which interactors aresought is purvalanol B, and the P2 are effectively a protein library,namely cellular extracts obtained from cultured Jurkat cells. The hybridligand as provided by the present invention, MTX-(CH₂—O—CH₂)₅-purvalanolB was synthesized as described in Example 1 above.

[0566] The assay described here somewhat follows the concepts developedin Knockaert et al. (2000), Chem. Bio. 7:411-422. Therein, purvalanol B,an inhibitor of CDKI/cyclinB, was immobilized on an agarose matrix andincubated with cellular extracts to identify polypeptides selectivelybinding to purvalanol B.

[0567] Pull down of DHFR-Hybrid Ligand-Interactor Complexes usingStreptactin Coated Resin

[0568] To construct a vector expressing a strep-tagged DHFR, the vectorpQE-40 (Qiagen, Hilden, Germany), which encodes a his₆-DHFR fusionprotein, under the expression-control of lac 0, upstream of a multiplecloning site comprising BglII and HindIII restriction sites, wasdigested to completion with BglII and HindIII (New England Biolabs,Beverly, Mass., USA) following manufacturer's protocols, andpurification was performed using a HiSpeed Tip and QiaPrecipitator(Qiagen, Hilden, Germany) according to manufacturer's instructions.

[0569] 50 μl each of 1 μM solutions of the 5′-phosphorylatedoligonucleotides ON284 and ON285 of sequences ON284 5′-GAT CTT GGA GCCACC CGC AGT TCG AAA AAT A-3′ (SEQ. ID No. 1) ON285 5′-AGC TTA TTT TTCGAA CTG CGG GTG GCT CCA A-3′ (SEQ. ID No. 2)

[0570] were combined and annealed by heating to 80° C. for 10 min andthen cooling continuously to 30° C. over 20 min. After annealing, thedouble stranded fragment possesses ends suitable for recombination withthe BglI and HindIII restriction ends of the linearized fragment ofpQE40, and, when ligated, encodes a strep tag fused in frame to thehis₆-DHFR fusion protein of pQE40.

[0571] The two fragments were combined and ligated using the QuickLigation Kit (New England Biolabs, Beverly, Mass., USA) according tomanufacturer's instructions. The purified vector was transfected into E.coli strain JM1O9 (Genotype: el4-(McrA-) recAl endAl gyrA96 thi-1hsdR17(rK− mK+) supE44 re1A1 Δ(Iac-proAB) [F′ traD36 proAB lacI^(q)ZΔM15]) (Stratagene, Amsterdam, Netherlands), over-expressing the lacIrepressor, by electroporation using standard procedures. Transformantswere cultured overnight under vigorous shaking at 200 rpm, 37° C. in LBmedium supplemented with 100 μg/ml ampicillin (LBAmp). This pre-culturewas adjusted with fresh LBAmp to an A600 nm=0.1, expression was inducedby adding isopropyl-PD-thiogalactoside (IPTG) to a concentration of 1mM, and the bacteria were allowed to grow for an additional 4 h. Cellswere harvested by 15 min centrifugation at 4500×g at 4° C. The resultingcell pellets were stored at −20° C.

[0572] 1 g of frozen cell pellets was re-suspended in 20 ml lysis-buffer(100 mM Tris, pH 8 containing 1 mg/ml lysozyme, 2 μg/ml avidin,10,000-fold diluted benzonase and protease inhibitors (P 2714, Sigma,Taufkirchen)) and sonicated for 10 min (pulse: 10 sec on; 10 sec off) at4° C. The crude extract was cleared by centrifugation at 10.000×g for 20min to remove cell debris and the strep-tagged protein products isolatedusing Strep-tactin Macroprep (IBA GmbH, Gottingen, Germany) affinitychromatography columns according to manufacturer's instructions.

[0573] Jurkat cells (DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Braunschweig, Germany; Cat. # ACC282) were culturedin 90% RPMI 1640 (Invitrogen, Carlsbad, Calif., USA, Cat. #42401042)+10% FBS (BioWhittaker, Verviers, Belgium, Cat. # US14-501 F)+2mM L-glutamine (Invitrogen, Carlsbad, Calif., USA, Cat. #25030024), seedout after thawing at about 1×10⁶ cells/ml, split ratio of about 1:2 to1:3 every 2-3 days; incubation at 37° C. with 5% CO₂). 2,5×10⁷ cellswere harvested upon attaining a cell density of approximately 1.5×10⁶cells/ml, pelleted at 300 g for 6 min at room temperature, and thepellet washed once with ice cold Dulbecco's PBS (PAA Laboratories, Linz,Austria, Cat. # H15-002). The pellet was re-suspended in 1 ml lysisbuffer: 150 mM NaCl, 15 mM MgCl₂ (Merck, Darmstadt, Germany, Cat. #1.06404.1000 and 1.05833.0250), 5 mM EDTA, 5% Glycerol (Serva,Heidelberg, Germany, Cat, # 11280 and 23176), 50 mM HEPES, pH 7.5, 1%Triton X-100, 1 mM DTT, 5 mM NaF, {fraction (1/10)} protease inhibitorcocktail and phosphatase inhibitor cocktail II (Sigma-Aldrich, St.Louis, Mo., USA, Cat. # H9897, T8787, D0632, S7920, P2714 and P5726).Cells were lysed at 4° C. for 20 min. After centrifugation for 15 min at4° C. and 16.000×g to remove cell debris, the pellet was discarded andthe supernatant stored at −80° C. for further use.

[0574] Strep-tactin Macroprep resin (IBA GmbH, Gottingen, Germany) wasalso used as the matrix resin for the pull-down methods. The matrix wasequilibrated with 30 volumes of chilled equilibration buffer (100 mMTris buffer, pH 8) for 30 min, the buffer decanted, and a slurry wasprepared by combining equal volumes of the wet resin and equilibrationbuffer.

[0575] 25 μl of his₆-DHFR-Strep, 1 mg/ml, were preincubated on ice with0.625 μl 20 mM NADPH and 1.25 μl 1 mM solution of the hybrid ligandMTX-(CH₂—O—CH₂)₃-purvalanol B for 15 min. Then 180 μl of Jurkat celllysate, corresponding to lysate from 4.5×10⁶ cells, was added. After 1hour incubation at 4° C. under tilt rotation 50 μl of theStreptactin-resin slurry were supplemented and again incubated for 1hour at 4° C. under tilt rotation. The resin was allowed to settle, thesupernatant removed carefully using a pipet, and the remaining resinwashed with 4×1 ml of ice-cold wash buffer (100 mM Tris, pH 8, 500 mMNaCl).

[0576] In order to elute the complexes bound to the resin, the resin isincubated 3 times with 36 μl of elution buffer (100 mM Tris, pH 8, 5 mMbiotin) for 10 min by resuspending the matrix several times, allowingthe matrix to settle, and removing the supernatant carefully using apipet. The supernatant fractions and the used resin were stored at −80°C. for further analysis.

[0577] For the identification of proteins that had bound toMTX-(CH₂—O—CH₂)₃-purvalanol B, the supernatant fractions were digestedwith trypsin, analysed by LC-mass spectrometry (LC: Altimate NanoLC,Dionex, Sunnyvale, Calif., USA; MS: Esquire 3000, Bruker Daltonics,Billerica, Mass., USA) and the results compared to a peptide fragmentlibrary. Essentially, the results presented in Knockaert et al. (2000),Chem. Bio. 7:411-422, were confirmed, as CDKl/cyclin B, CDK2/cyclin A,CDK5/p25, CK1 (mammalian), Erkl, Erk2, S6 kinase (p7orsk) and CaM kinaseII were identified.

Example 4 Pull-Down of Polypeptides Bound to the DNA Encoding them whichInteract with a Hybrid Ligand of the Invention

[0578] While pull-down experiments represent a useful method to isolatepolypeptides binding to or interacting with a molecule of interest, thedirect identification of polypeptides, for example using massspectrometry or amino acid sequencing, is more cumbersome and lessreliable than DNA sequencing. WO 93/08278, WO 98/37186, WO 01/14539 andWO 02/22826 present methods which combine the relative ease of pull-downmethods in isolating molecular species interacting with a given targetmolecule with the convenience of DNA sequencing for the identificationof polypeptide interactors.

[0579] In order to demonstrate the applicability of the methods shown inWO 93/08278, WO 98/37186, WO 01/14539 and WO 02/22826 to the hybridligands of the present invention, we devised an experimental protocolwherein a cDNA-library is expressed as a library of fusion proteinscomprising a DNA-binding domain. The vectors encoding the library offusion proteins each comprise the corresponding DNA-motif which theDNA-binding domain binds to. When expressed in a suitable host, thefusion proteins will bind to the vectors encoding them, and thecomplexes may be isolated and used for panning. For the panning ofinteraction partners for the hybrid ligands of the invention of generalstructure R1-Y-R2, the known interactor for R1, P1, is immobilized on amatrix, and the hybrid ligand is added such that it will be attached tothe matrix via its binding to P1. Subsequently, the DNA-polypeptidecomplexes are added and unbound complexes are washed away. Finally, theDNA is isolated from the complexes and sequenced.

[0580] Construction of Plasmids pMC3 and pMC520

[0581] The bacterial strains used are E. coli K12 strains MC1061(araD139 Δ(araABC-leu)7696 thr ΔlacX74_ga1U ga1K hsd.R mcrB rpsL(strA)thi), AR120 (F′ lac⁺ pro⁺ lacIq18 laclam74//Δ (lac-pro) thi rpsL(strA)recA::cat), and XL1-Blue (F′ proAB lacIq lacZΔM15 Tn10// recA1 endA1gyrA96 thi hsdR17 supE44 re1A1 lac), and E. coli B strain ARI 161(ion-11, su1A1, hsdR17, Δ(ompT-fepC), ΔclpA319::kan). AR1161 is aprotease deficient strain and serves to minimize proteolysis of thepeptides in the library, which would reduce the available diversity forpanning. Mutations known to reduce proteolysis include degP, Ion, htpR,ompT, and clpA,P.

[0582] The library plasmid pMC5 is constructed in several steps usingplasmid pBAD18 as the starting plasmid. Plasmid pBAD18 contains the araBpromoter followed by a polylinker and a terminator under the control ofthe positive/negative regulator AraC, also specified by the plasmid.Plasmid pBAD18 also contains a modified plasmid pBR322 origin and thebla gene to permit replication and selection in E. coli, as well as thephage M13 intragenic region to permit rescue of single-stranded DNA forsequencing.

[0583] The lacI gene is modified for cloning into plasmid pBAD18 usingthe GeneAmp® PCR amplification kit (Perkin-Elmer Cetus Instruments) witholigonucleotides ON-286 and ON-287, shown below: ON-286 5′-GCG GGC TAGCTA ACT AAT GGA GGA TAC ATA AAT (SEQ. ID No. 3) GAA ACC AGT AAC GTT ATACG-3′ ON-287 5′-CGT TCC GAG CTC ACT GCC CGC TCT CGA GTC GGG (SEQ. ID No.4) AAA CCT GTC GTG C-3′.

[0584] The amplification reaction is carried out according to themanufacturer's instructions, except for the use of Vent™ DNA polymerase(New England Biolabs, Beverly, Mass., USA). ON-286 contains anonhomologous 5′ region that adds an NheI site, a consensus ribosomebinding site (see Gold and Stormo, 1990, Methods in Enzymology (Goeddel,ed., Boston: Academic Press), pp. 89-103, incorporated herein byreference), and changes the initiation codon of lacI from GTG to ATG.ON-287 changes codons 356 and 357 of lacI to an XhoI site through twosilent mutations, and adds a SacI site after the lacI stop codon. Thesequence of the PCR product is confirmed by sequencing.

[0585] Cloning of the NheI, SacI digested amplification product intoplasmid pBAD18 produced vector pJS100. Two lacO_(s) sequences are addedto this vector, with their centers spaced 326 bp apart, by amplifying anunrelated sequence (the human D2 dopamine receptor gene; see: England etal., 1991, FEBS Lett. 279:87-90, incorporated herein by reference), witholigonucleotides ON-295 and ON-296, shown below: ON-295 5′-CCT CCA TATGAA TTG TGA GCG CTC ACA ATT CGG (SEQ. ID No.5 TAC AGC CCC ATC CCA CCC-3′ON-296 5′-CGC CAT CGA TCA ATT GTG AGC GCT CAC AAT TCA (SEQ. ID No. 6)GGA TGT GTG TGA TGA AGA-3′

[0586] ON-295 adds an NdeI site and a lacO_(s) sequence at one end ofthe amplified fragment, and ON-296 adds a ClaI site and lacO_(s) at theother end. Cloning of the NdeI to ClaI fragment into pJS100 producedplasmid pJS102.

[0587] Plasmid pMC3, encoding the dynorphin B-tailed lac repressor, isconstructed by cloning complementary oligonucleotides ON-312 and ON-313to replace the XhoI to XbaI fragment at the 3′ end of lacI in pJS102.These oligonucleotides add sequence encoding a five amino acid spacer(GADGA (SEQ. ID No. 7)) and dynorphin B (YGGFLRRQFKVVT (SEQ. ID No. 8))to the end of the wild-type lacI sequence, introduce an SfiI site in thesequence encoding the spacer, and are shown below: ON-312 5′-TCG AGA GCGGGC AGG GGG CCG ACG GGG CCT ACG (SEQ. ID No. 9) GTG GTT TCC TGC GTC GTCAGT TCA AAG TTG TAA CCT AAT-3′ ON-313 5′-CTA GAT TAG GTT ACA ACT TTG AACTGA CGA CGC (SEQ. ID No. 10) AGG AAA CCA CCG TAG GCC CCG TCG GCC CCC TGCCCG CTC-3′

[0588] The library plasmid pMC5 is constructed by cloning complementaryoligonucleotides ON-335 and ON-336 to replace the SfiI to HindIIIdynorphin B segment of pMC3. Oligonucleotides ON-335 and ON-336 areshown below: (SEQ. ID No. 11) ON-335 5′-GGG CCT AAT TAA TTA-3′ (SEQ. IDNo. 12) ON-336 5′-AGC TTA ATT AAT TAG GCC CCG T-3′

[0589] Plasmid pMC3 is available in strain ARI161 from the American Typeculture Collection under the accession number ATCC No. 68818.

[0590] Construction of a cDNA Library in Vector pMC5

[0591] To construct a library of plasmids encoding the DNA-binding lacrepressor fused to a library of polypeptides, a cDNA library isgenerated from poly A+ RNA isolated from human fetal brain (hFB)(Clontech, CAT# 6525-1) essentially using a commercially availableprotocol and reagents (Superscript, Invitrogen, Carlsbad, Calif., USA,CAT. NO. 18248-013) but employing oligo-dT primers for first-strandsynthesis as follows: TT2-A: 5′TTT TGT ACA TCT AGA TCG CGA AAG CTT CTTTTT TTT (SEQ. ID No. 13) TTT TTT TV-3′

[0592] with V being A, G, or C at equal molar ratio. This primerintroduces a HindIII restriction site.

[0593] The 3′-phosphorylated oligonucleotides ON 369 and ON 370 aresynthesized to possess the sequences ON 369: 5′-AGC AGC AGC AGC-3′ (SEQ.ID No. 14) ON 370 5′-GCT GCT GCT GCT GCT-3′ (SEQ. ID No. 15)

[0594] When annealed and ligated to the cDNA library, theseoligonucleotides create an end compatible with the SfiI-restrictionfragment of vector pMC5.

[0595] 400 pmol of each oligonucleotide are annealed in 25 μl reactionbuffer (10 mM Tris, pH 7.4, 1 mM EDTA, 100 mM NaCl), by heating to 65°C. for 10 min and cooling for 30 min to room temperature, and ligated tothe cDNA-library by standard procedures. The vector pMC5 is digested tocompletion with SfiI and HindIII, the cDNA library only with HindIII,and the vector backbone and the cDNA digestion products are isolated by4 rounds of washing with TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) in aCentricon 100 microconcentrator (Amicon) by the manufacturer'sinstructions, followed by phenol extraction and ethanol precipitation.The digested library is added to 64 μg of digested pMC5 in a 3.2 mlligation reaction containing 5% PEG, 3200 units of HindIII, 194 Weissunits of T4 ligase (New England Biolabs, Beverly, Mass., USA), 1 mM ATP,20 mM Tris, pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 50 μg/ml BSA, and 2 mMDTT. The reaction is split equally into 8 tubes and incubated overnightat 15° C.

[0596] After ethanol precipitation, {fraction (1/16)} of the ligated DNA(4 μg) is introduced into MC1061 (80 μl) by electroporation (Dower etal., 1988, Nucl. Acids Res. 16:6127-6145, incorporated herein byreference), to yield 5.5×10⁸ independent transformants. The library isamplified approximately 1000-fold in 1 liter of LB/100 μg/ml ampicillinby growth of the transformants at 37° C. to an A₆₀₀ of 1. The cellscontaining the library are concentrated by centrifugation at 5500×g for6 min, washed once in ice-cold 50 mM Tris (pH 7.6), 10 mM EDTA, 100 mMKCl, followed by a wash in ice-cold 10 mM Tris, 0.1 mM EDTA, 100 mM KCl.The final pellet is resuspended in 16 ml of HEG buffer (35 mM HEPES/KOH,pH 7.5, 0.1 mM EDTA, 100 mM Na Glutamate), distributed into 19 tubes of1.0 ml each, frozen on dry ice, and stored at −70° C.

[0597] Panning the Library

[0598] Vector pQE40 (Qiagen, Hilden, Germany), comprising the geneencoding DHFR fused to a his6-tag, is transformed into E. coli strainJM109 and the His₆-DHFR fusion protein isolated and purified asdescribed above (see Example 3). His₆-DHFR is subsequently coupled toHIS-Select HS Nickel coated microtiter plates (Sigma, Cat. No. S 5688)according to manufacturers instructions using a volume of 150 μl/well ata concentration of 1,5 μg/ml His₆-DHFR fusion protein in PBS (Sigma,Cat. No. P 3813) at pH 7.0 for 4 h at room temperature. Wells are washed3 times with PBS containing 0,05% Tween 20 (Sigma, Cat. No. P 3563),followed by incubation with 100 μl] of a 10 uM solution of GPC 285985 inPBS, pH 7.0, containing 0,5 mM NADPH, for 1 h at room temperature, orwith 0,5 mM NADPH PBS for control wells.

[0599] One aliquot (1.0 ml) of the library prepared in Example 2 isthawed on ice and added to 9 ml of lysis buffer (35 mM HEPES adjusted topH 7.5 with KOH, 0.1 mM EDTA, 100 mM Na glutamate, 5% glycerol, 0.3mg/ml BSA, 1 mM DTT, and 0.1 mM PMSF). Lysozyme is added (0.3 ml at 10mg/ml in HEG), and the mixture is incubated on ice for 1 hr.

[0600] The cellular debris is removed by centrifugation of the lysate at20,000×g for 15 min, and the supernatant is concentrated bycentrifugation in a Centriprep® 100 concentrator (Amicon) at 500×g for40 min The concentrated supernatant (about 0.5 ml) is washed with 10 mlof HEG buffer and centrifuged as before. A sample (5%) of the totallysate is removed to determine the pre-panned input of plasmidcomplexes.

[0601] An alternate method for partially purifying and concentrating thelysate is as follows. About 2.0 ml of the frozen cells in HEG are thawedon ice, and then 8 ml of lysis buffer without Na glutamate (high ionicstrength inhibits lysozyme; DTT is optional) are added to the cells, andthe mixture is incubated on ice for 1 hr. The cellular debris is removedfrom the lysate by centrifugation at 20,000×g for 15 min, and thesupernatant is loaded onto a Sephacryl® S-400 High Resolution(Pharmacia) gel-filtration column (22 mm×250 mm). The plasmid-fusionprotein complexes elute in the void volume. The void volume (30 ml) isconcentrated with two Centriprep® 100 concentrators, as described above.After adjusting the Na glutamate concentration of the concentrate, onecarries out the remainder of the procedure in the same manner as withthe first method.

[0602] 195 μl of the concentrated lysate are adjusted to 0,5 mM NADPH byadding 5 μl of 20 mM NADPH in PBS, and the solution is added to aDHFR-GPC 285985 treated well of a HIS-Select HS Nickel coated microtiterplate, another 200 μl of this solution is added to a control welllacking GPC 285985. After incubating the lysates in the wells at 30° C.for 1 hr. with shaking, the wells are washed three times with 200 μl ofcold HEG/O. 1% BSA and then three times with HEG. The plasmids aredissociated from the wells by phenol extraction, and after adding 20 pgof glycogen 35 (Boehringer Mannheim), the DNA is precipitated with anequal volume of isopropanol. The pellet is washed with 75% ethanol, andthe DNA is resuspended in 4 μl of H₂O. Strain MC 1061 is transformedusing 2 μl each of the DNA solutions to permit counts of recoveredplasmids and amplification of the selected plasmids. Optionally,additional rounds of panning may be added.

[0603] ELISA Analysis of the Library

[0604] An ELISA is used to test MC1061 transformants from the panningfor GPC 285985-specific ligands. The ELISA is performed in a 96-wellplate (Beckman). Single colonies of transformants obtained from panningare grown overnight in LB/100 μg/ml ampicillin at 37° C. The overnightcultures are diluted {fraction (1/10)} in 3 ml LB/100 μg/ml ampicillinand grown 1 hr. The expression of the lac repressor-peptide fusions isinduced by the addition of arabinose to a final concentration of 0.2%.

[0605] The cells are lysed as described above in 1 ml of lysis bufferplus lysozyme and stored at −70° C. Thawed crude lysate is added to eachof 2 wells (1.00 μl/well), and the plate is incubated at 37° C. After 45min, 100 μl of 1% BSA in PES (10 mM NaPO4, pH 7.4, 120 mM NaCl, and 2.7mM KCl) are added for an additional 15 min at 37° C., followed by 3washes with PBS/0.05% Tween 20. Each well then is blocked with 1% BSA inPES (200 μl/well) for 30 min at 37° C., and the wells are washed asbefore.

[0606] GPC 285985 (100 μl of a 10 μM solution in PBS/0.1% BSA) is addedto each well, the plate is incubated at room temperature for 1 hr., andthen each well is washed as before. His₆-DHFR fusion protein is preparedas described above, and 150 μl/well at a concentration of 1,5 μg/mlHis₆-DHFR fusion protein in PBS/0.1% BSA is added to the wells andincubated for 2 h at 37° C., followed by 3 washes with 150 μl PBS/0.1%BSA. Bound His₆-DHFR fusion protein is detected using an anti-His6antibody coupled to horseradish peroxidase (RGS-His HRP Conjugate Kit,Qiagen, Hilden, Cat. # 34450) following manufacturers instructions. Todetermine the structure of the peptide ligands obtained by the presentmethod, plasmids from both ELISA positive and ELISA negative coloniesobtained after panning are sequenced. Double stranded plasmid DNA,isolated from strain XL1-Blue, is sequenced using Sequenase@ (USBiochemicals) according to the instructions supplied by themanufacturer.

Example 5 Construction of Genetic Constructs and Yeast Strains for aYeast Three Hybrid Experiment Employing a Transcriptional-BasedInteraction System

[0607] A yeast three hybrid experiment employing a transcriptional-basedinteraction system was demonstrated by utilizing a yeast straincomprising three genetic constructs: a first construct encoding a fusionprotein comprising a DNA-binding domain (BD) and a first protein orpeptide (P1) able to specifically bind the first ligand R1 of theenvisaged hybrid ligand R1-Y-R2; a second construct encoding a fusionprotein comprising a transcriptional activation domain (AD) and a secondprotein or peptide, or a library of second proteins or peptides, (P2)able or suspected to bind the second ligand R2 of said envisaged hybridligand; a third construct comprising a reporter gene under thetranscriptional control of a promoter comprising the genetic sequencethe BD is able to bind to, wherein the AD must be capable of initiatingthe transcription of the reporter gene when brought in spatial proximityof the promoter via bridging interaction of the hybrid ligand betweenthe BD-comprising fusion protein and the AD-comprising fusion protein.

[0608] Two plasmids were constructed: the first plasmid containing afragment encoding the bacterial LexA binding domain for expression as afusion with a first protein; the second plasmid containing a fragmentencoding the yeast GAL4 transcriptional activation domain for expressionas a fusion with a second protein. These plasmids were transformed intoyeast cells deficient in the endogenous HIS3 locus but comprising agenetic construct combining a recombinant his3 gene with a promotercontaining the LexA binding sequence. Since methotrexate was chosen asthe first ligand R1 in the present investigations, the sequence encodingthe LexA BD was fused to the gene encoding E. coli dihydrofolatereductase (folA). The sequence encoding the GAL4 transcriptionalactivation domain was fused either to the gene encoding thedexamethasone-binding rat glucocorticoid receptor gr2, the genes forhuman cdk2 (hcdk2) or cdk4 (hcdk4) or to a library of genes from a humanbrain cDNA library, depending on the choice of R2.

[0609] Yeast strain L40 (Invitrogen, Carlsbad, Calif., USA,; MATa,his3-A200, trp1-901, leu2-3,112, ade2, LYS2::(1exAop)₄—HIS3,URA3::(1exAop)₈-LacZ, ga180) was chosen for the experiments in yeastsdescribed herein. However, other suitable yeast strains, or even othercell types, such as bacteria, insect cells, plant cells or mammaliancells may be chosen for the methods of the invention, provided, thecells comprise a reporter system that allows a detectable readout thatis conditional on the formation of a trimeric complex of the hybridligand together with the first and second fusion proteins.

[0610] For the DNA binding domain-fusion plasmid, the E. coli folA(dihydrofolate reductase, DHFR) coding sequence was PCR amplified from agenomic library (Clonetech, Cat. No.: XL4001AB) using primers CR89 andCR 90 CR 89 5′-GGG GTC GAC ATG ATC AGT CTG ATT GCG GCG TTA (SEQ. ID No.16) GCG-3′ CR 90 5′-GGG GGC GGC CGC TTA CCG CCG CTC CAG AAT CTC (SEQ. IDNo. 17). AAA G-3′

[0611] The sequence of the PCR product was confirmed by sequencing. ThePCR product was digested with SalI and NotI, and the resulting 479 bpfragment was subcloned into pBTM118c containing TRP1 as a selectablemarker in yeast (see Wanker et al., WO 99/31509), resulting in theconstruct pBTM 118c-DHFR.

[0612] For the activation domain fusion-plasmid comprising the ratglucocorticoid receptor, a gene fragment encoding amino acids 524-795 ofthe rat glucocorticoid receptor was PCR amplified from a rat brain cDNAlibrary (Life Technologies, Cat. No. 10653-012) using primers CR91 andCR92: CR91 5′-GGG GTC GAC ATG GGT GGT GGT GGT GGT GGT GCA (SEQ. ID No.18) GGA GTC TCA CAA GAC-3′ CR92 5′-GGG GGC GGC CGC TTT TTG ATG AAA CAGAAG-3′. (SEQ. ID No. 19)

[0613] The sequence of the PCR product was confirmed by sequencing. ThePCR product was digested with SalI and NotI, and the resulting 813 bpfragment was subcloned into pGAD426c containing LEU2 as a selectablemarker in yeast (Wanker et al., WO 99/31509). Subsequently, amino acidsF620 and C656 of GR2 were replaced with Ser and Gly respectively toincrease the affinity of GR2 for dexamethasone (Chakraborti et al.,1991, J. Biol. Chem., 266: 22075-22078), using a site-directedmutagenesis PCR reaction. Mutagenesis was performed employing the“QuickChange Site directed mutagenesis kit” (Stratagene, Amsterdam,Netherlands) according to manufacturers protocols. The presence of thesemutations was confirmed by sequencing. The resulting construct wasdesignated pGAD426c-GR2.

[0614] For the activation domain fusion comprising hcdk2, the cDNAencoding hCDK2 was amplified from the human placenta MATCHMAKER cDNAlibrary (C!ontech, Cat# HL4025AH, Heidelberg, Germany) by PCR usingprimers CR92 and CR93 (SEQ. ID No. 20) CR92 5′-GGG TCG ACG CAT GGA GAACTT CC-3′ (SEQ. ID No. 21) CR93 5′-GGG CGG CCG CTC AGA GTC GAA G-3′.

[0615] Similarly, hcdk4 cDNA was amplified by PCR using primers CR94 andCR95: (SEQ. ID No. 22) CR94 5′-GGG TCG ACG CAT GGC TAC CTC TCG-3′ (SEQ.ID No. 23) CR95 5′-GGGCGGCCGCTCAGGCTGTATTCAGC-3′.

[0616] The sequences of the PCR products were confirmed by sequencing.After digestion of the PCR products with SalI and NotI, the resulting894 bp (CDK2) and 909 bp (CDK4) fragments were individually subclonedinto pGAD426c, and the sequences of the clones verified by DNAsequencing. The resulting constructs were termed pGAD426c-hCDK2 andpGAD426c-hCDK4, respectively.

[0617] A library of human fetal brain cDNA's fused to the gene encodingthe GAL4 activation domain cloned into vector pACT2 (Clontech, Cat. No.:HY4004AH; see FIG. 17) bearing LEU2 as a yeast selectable marker wasused as purchased for clone selection experiments in yeast as describedin Example 10.

Example 6 The Halo Growth Assay

[0618] A halo growth assay was conducted to test the dimerizing capacityof hybrid ligands of the invention. FIG. 4 a. shows a halo growth in apetri dish spotted with GPC 285937. Dimerization of the LexA-DNA BindingDomain (LexA-BD)—DHFR and GAL4-transcription activation domain(GAL4-AD)GR2 fusion proteins in the presence of GPC 285937 in the L40yeast strain caused transcription of the His3 reporter gene. Thistransciptional expression of HIS3 enabled the yeast cells to overcomethe lack of histidine in the medium, leading to cell growth in the areato which sufficient GPC 285937 had diffused from the center of the dish.Conversely, no visible growth appears in the control dish spotted withDMSO only shown in FIG. 4b.

[0619] To conduct the halo assay, plasmids pGAD426c-GR2 andpBTM118c-DHFR were co-transformed into the yeast strain L40 usingstandard yeast methods (Burke at al., Methods in yeast genetics: A ColdSpring Harbor Laboratory course manual; Cold Spring Harbor LaboratoryPress, 2000). Transformants receiving both plasmids were selected onmedia lacking trp and leu. Individual colonies were then inoculated andincubated in liquid SD-medium for 24 hrs. The cultures were diluted to adensity of 10⁶ cells/ml and 100 μl were plated on a 10 cm petri dishcontaining SD medium lacking trp, leu and his. 1 μl of a 1 mM solutionof GPC 285937 dissolved in DMSO or 1 μl of DMSO as control was spottedin the center of each petri dish. The growth of yeast cells wasdetermined after 2 days of growth at 30° C.

Example 7 The Fluorescence Detection Growth Assay

[0620] To demonstrate the suitability of the fluorescence detectiongrowth assay employing the PreSens Precision Sensing GmbH (Regensburg,Germany) OxoPlate, an experiment analogous to Example 4 was performed.Yeast cells were transformed with the plasmid encoding the DHFR-LexA DNAbinding domain fusion protein and either the plasmid encoding hCDK2 orhCDK4 fused to the GAL4 activation domain. Cells of the resulting strainwere seeded into wells of an Oxoplate and exposed to one of fourconditions: 1) SD medium lacking leu and trp (positive control); 2) SDmedium lacking leu, trp and his (negative control); 3) SD medium lackingleu, trp and his and supplemented with a range of concentrations (1 mMto 4 μM) of GPC 285985, a compound known to bind strongly to DHFR andhCDK2, but only weakly to hCDK4; 4) SD medium lacking leu, trp and hisand supplemented with 1 mM GPC 285993, a compound known to bind stronglyto DHFR, but not to hCDK2 or hCDK4 (compound selectivity control).

[0621] The results obtained in this experiment are represented in FIG.8, and as expected, no oxygen consumption due to growth of cells wasobserved in the negative controls or the compound selectivity controls.In contrast, growth was observed in the positive controls and in thecells transformed with the construct encoding the hCDK2 fusion proteinat all concentrations of GPC 285985, albeit growth onset was slightlydelayed at the lowest concentrations of GPC 285985. Cells transformedwith the construct encoding the hCDK4 fusion protein grew only whenexposed to a high concentration (1 mM) of GPC 285985, further confirmingthe specificity of binding of this hybrid ligand compound to hCDK2.

[0622] The fluorescent assay was conducted as follows: First, cells ofyeast strain L40 were co-transformed with pBTM 118c-DHFR and one ofeither pGAD426c-hCDK2 or pGAD426c-hCDK4 using standard techniques (Burkeat al., Methods in yeast genetics: A Cold Spring Harbor Laboratorycourse manual; Cold Spring Harbor Laboratory Press, 2000). Transformantscontaining both plasmids were selected on SD medium lacking trp and leu,and individual colonies were inoculated in liquid SD-medium andincubated for 48 hrs at 30° C. Second, cells were precipitated andwashed with sterile water 3 times, the cell number adjusted to a densityof 10⁸ cells/ml and 50 μl transferred to each well of an OxoPlate F96(PreSens Precision Sensing GmbH, Regensburg). 150 μl of a solutionrepresenting one of four conditions was added: 1) SD-medium lacking leu,trp and his (wells Al1-F1, negative control); 2) SD -leu, -trp (wellsA2-F2, positive control), 3) SD-medium lacking leu, trp and hissupplemented with the compound GPC 285985 at concentrations of 1 mM, 0,5mM, 0,25 mM. 125 μM, 63 μM, 31 μM, 16 μM, 8 μM or 4 μM (wells A3-F11);4) SD-medium lacking leu, trp and his supplemented with 1 mM of thecontrol compound GPC 285993 (A12-F12, compound selectivity control).Third, oxygen consumption of growing yeast cells was monitored as afunction of the ratio of fluorescent emissions of a first fluorescentdye that was quenchable by oxygen (emission at 590 nm) and a second dyeunquenchable by oxygen (emission at 640 nm). This ratio of fluorescencewas monitored over 18 hours in 20 min intervals at 30° C. using a PerkinElmer Wallac Victor2 V 1420 multilabel HTS counter (Perkin Elmer,Wellesley, Mass., USA) with an excitation setting of 540 nm and anemission setting of 590/640 nm (dual kinetic mode).

Example 8 Testing of Hybrid Ligand Compounds for Effects not Related toDimerization

[0623] Effects of hybrid ligand compounds independent of theirdimerizing action on the cells used for an assay may invalidate resultsfrom assays employing these compounds. Such effects may be, for example,toxicity or growth promotion via routes other than lack of, or inducedproduction of, leucine, tryptophane and/or histidine in the assaysdescribed above. Therefore, the in vivo effect of the hybrid ligands wasdetermined in a halo growth assay as described in Example 4, but usingempty (i.e. not containing the subcloned gr gene and hence lacking asecond ligand P2 to bind R2) pGAD426c instead of pGAD426c-GR. 1 μl eachof a dilution series of the hybrid ligands (10 mM to 1 μM in DMSO) wereused for spotting in the center of petri dishes prepared to containeither medium lacking trp and leu, or trp, leu and his and plated withL40 yeast cells containing the plasmids pGAD426c and pBTM118c-DHFR.Growth was monitored after two days of incubation at 30° C. Cells areexpected to grow irrespective of concentration of the hybrid ligandcompound on media lacking only trp and leu, while no growth shouldappear on media lacking trp, leu and his. This expected behaviour wasobserved with all hybrid ligand compounds used herein at allconcentrations tested.

Example 9 Improved Functionality of the Dimerizing Hybrid Ligands of thePresent Invention over the State of the Art

[0624] To compare Mtx-mdbt-Dex (Lin et al., J. Am. Chem. Soc. 2000,122:4247-8) with Mtx-(ethylenglycol)₃-Dex (GPC 285937) in a yeast threehybrid assay, we first prepared dilutions of both compounds in liquid SDmedium lacking his, trp and leu, in a concentration range from 1 mM to 1μM by adding the appropriate amount of compound dissolved in DMSO to themedium. Second, L40 yeast cells were transformed with plasmids pBTM118c-DHFR and pGAD426c-GR2 and inoculated into the media containing thecompounds in different amounts at a density of 0.1 OD₅₉₅. Growth wasmonitored for 48 hours by measuring OD₅₉₅ on a Perkin Elmer WallacVictor2 V 1420 multilabel HTS counter (Perkin Elmer, Wellesley, Mass.,USA). It appeared that the yeast strain grew in a window of between 25to 400 μM showing optimum growth at 100 μM GPC 285937 (Data not shown).However, at these concentrations, Mtx-mdbt-Dex showed severeprecipitation in the medium (See FIG. 5). This precipitation may causethe compound to be less bio-available and hence growth of yeast cells inthe presence of this compound to be impaired.

[0625] The functional advantages of a hybrid ligand of the invention;Mtx-(ethylenglycol)₃-Dex (GPC 285937) over the prior-art compoundMtx-mdbt-Dex was further shown in a halo assay as follows. First, L40yeast strain was transformed with plasmids pBTM118c-DHFR andpGAD426c-GR2 and transformants containing both plasmids were selected onmedia lacking trp and leu. Second, individual colonies were inoculatedin liquid SD-medium and incubated for 24 hrs. The cell cultures werediluted to a density of 106 cell/ml and 100 μl were plated on a 10 cmpetri dish containing SD medium lacking trp, leu and his. Third, 1 μl ofa 1 mM solution of GPC 285937 (three ethylenglycol units as linker) orMtx-mdbt-Dex (metadibenzothioester as a linker) dissolved in DMSO wasspotted in the center of each petri dish. The growth of yeast cells wasdetermined after 2 days of growth at 30° C.

[0626]FIG. 6 a. shows the growth halo that developed around the point ofapplication of GPC 285937, while FIG. 6b displays the same result forMtx-mdbt-Dex. The growth halo of yeast cells receiving Mtx-mdbt-Dex wasmuch smaller than that of the hybrid ligand of the invention, furtherdemonstrating the superiority of the latter.

[0627] A hybrid ligand of the invention also showed significantimprovement over the prior art hybrid ligand under conditionsappropriate to library screening of yeast cells. The yeast strain L40was cotransformed with the plasmids pBTM 118c-DHFR and pGAD426c-GR2.Transformants containing both plasmids were selected on media lackingtrp and leu, and individual colonies were inoculated in liquid SD-mediumand incubated for 24 hrs. These cell cultures were diluted to a densityof 10⁴ cell/ml and 2×10⁴ cells were plated on 22×22 cm plates containingyeast synthetic agar medium lacking his, trp and leu but containing 200μM GPC 285937 or Mtx-mdbt-Dex. Growth of individual colonies wasmonitored after 48 h at 30° C. Colonies growing on SD-media withMtx-mdbt-Dex were hardly detectable, whereas clones visibly grew betteron media containing GPC 285937, a hybrid ligand of the invention (FIG.7).

Example 10 Advantages of Different Embodiments of the Dimerizing HybridLigands of the Present Invention

[0628] For certain small molecules, particular physiochemical propertiessuch as solubility may require a particular choice of linker to be usedin order to generate particularly advantageous hybrid ligands of thegeneral structure R1-Y-R2. For example, the bioavailability and, hence,biological activity may be further enhanced by adding additional(—CH2-X-CH2) repeats to the linker Y. This was the rationale behind thesynthesis of the hyrbid ligands GPC 286004 (comprising an(ethylenglycol)₃ linker and GPC 286026 comprising an (ethylenglycol)₅linker. Plasmid pGAD426c-hCDK2 was co-transformed with pBTM118c-DHFRinto the yeast strain L40. Transformants containing both plasmids wereselected on media lacking trp and leu, and individual colonies wereinoculated in liquid SD-medium and incubated for 24 hrs. These cultureswere diluted 1:10 and 20 μl of the diluted culture was spotted induplicate on a 10 cm petri dish containing SD medium that lacks trp, leuand his. 1 μl of a 1 mM solution of GPC 286004 or GPC 286026 dissolvedin DMSO was spotted in the center of each spot. The growth of yeastcells was determined after 3 days of growth at 30° C. The results ofthis halo assay show that after 3 days on medium lacking leu, trp andhis, halo growth was only seen in the presence of GPC 286026 (fiveethylenglycol units as linker; FIG. 16b.) but not in the presence of GPC286004 (three ethylenglycol units as linker; FIG. 16 a.), Thisdemonstrated the superior suitability of the (ethylenglycol)₅ linkergroup over the (ethylenglycol)₃ linker group when linking these twoparticular compounds to form a hybrid ligand.

Example 11 Methods of Testing a Polypeptide for Binding to aUser-Specified Ligand: a Three-Hybrid Assay System Based on a ReporterSystem Using Transcriptional Activation

[0629] In certain embodiments, the methods of the invention are used totest polypeptides for their ability to bind to a user-specified ligand.To demonstrate this concept, we first designed a three-hybrid experimentusing a small-molecule compound to distinguish between two polypeptides.The first polypeptide was known to bind with high affinity to thesmall-molecule compound, while the second polypeptide was known to bindto the small-molecule compound only weakly. For this purpose, saidsmall-molecule compound was integrated into a hybrid ligand of theinvention, and used in a three hybrid screen with atranscriptional-based interaction system.

[0630] A hydropyrazolo-pyrimidine-moiety was developed by GPC as aselective inhibitor of hCDK2. It binds with high affinity to hCDK2 butonly weakly to hCDK4 as can be determined for example using a methodanalogous to Example 4. When linked via a (—CH₂—O—CH₂)₃-linker toMethotrexate (GPC 285985), the resulting hybrid ligand should beexpected to bind to and bridge a combination of BD-DHFR and hCDK2-ADfusion proteins, and consequently activate a lexA-controlled reportergene. However, the same hybrid ligand should not be able to bind to andbridge the combination of BD-DHFR and hCDK4-AD fusion proteins when usedat working concentrations. To test this hypothesis, cells of yeaststrain L40 were co-transfected with pBTM 118c-DHFR and eitherpGAD426c-hCDK2 or pGAD426c-hCDK4 as appropriate. Transformants receivingboth plasmids were selected on media lacking trp and leu, and individualcolonies were inoculated in liquid SD-medium and incubated for 24 hrs.These two yeast strain cultures were diluted to a density of 106 cell/mland 100 μl of each diluted culture were plated on a 10 cm petri dishcontaining SD medium lacking trp, leu, and also on a 10 cm petri dishcontaining SD medium lacking trp, leu and his. 1 μl of a 1 mM solutionof GPC 285985 dissolved in DMSO or 1 μl DMSO as a control was spotted inthe center of each petri dish. The growth of yeast cells was determinedafter 2 days of growth at 30° C. (FIG. 10) where growth was seen onmedium lacking leu, trp and his only for cells containingpGAD426c-hCDK2. After 6 days, cells containing pGAD426c-hCDK2 hadcompletely overgrown the petri dish, while very minimal growth wasobserved in cells containing pGAD426c-hCDK4 (FIG. 11). This isconsistent with the relative affinities of GPC 285985 for hCDK2 andhCDK4, and demonstrates a method of testing the ability of a polypeptideto bind to a user-specified ligand.

Example 12 Methods of Identifying a Polypeptide that Binds to aUser-Specified Ligand: a Three-Hybrid Assay System Based on aTranscriptional-Based Interaction System

[0631] To demonstrate the suitability of certain methods of theinvention for the identification of polypeptides that bind to auser-specified ligand from large collections of candidate polypeptides,a genetic screen was carried out using three hybrid molecules: first,GPC 285985, a hybrid ligand of the invention; second, a BD-DHFR fusionprotein able to bind to the methotrexate moiety in GPC 285985 and bindto the lexA promoter; third, a library of human fetal brain cDNA's fusedto the GAL4-AD. As a negative control, an alternative hybrid hybridligand comprising a small molecule linked to methotrexate via a(—CH₂—O—CH₂)₃-linker so as to be unable to bind to hCDK2 (GPC 285993)was used to confirm compound specific growth.

[0632] The 3-hybrid screen of the invention was conducted as follows.First, cells from yeast strain L40 were transformed with pBTM 118c-DHFR,and transformants receiving the plasmid were selected on syntheticmedium lacking tryptophan. Second, individual colonies were regrown inliquid media, rendered competent and the L40 cells containingpBTM118c-DHFR were transformed with a human fetal brain cDNA librarycloned in vector pACT2 (Clontech, Cat. No: HY4004AH). 1×10⁷ individualcolonies were selected on 60 22×22 cm SD agar plates lacking trp andleu. After three days of growth at 30° C. the colonies were washed offthe plates, mixed and frozen in small aliquots. 2×10⁶ cells were platedon each of 18 SD plates containing media lacking trp, leu and his butcontaining 20 μM of GPC 285985 and incubated for 2-5 days. A total of2811 colonies appeared and were picked into 384 well microtiter platescontaining SD medium lacking trp and leu. All clones were tested in ahigh-throughput halo assay against GPC 285985 dissolved in DMSO asgrowth promoter, or GPC 285993 dissolved in DMSO, or pure DMSO (LTH) asnegative control. This halo assay was analogous to that described inExample 4 except that multiple different assays (between 10 and 1000)were tested in singular or replicate on 22×22 cm agar trays containingappropriate growth media. Test and control yeast strains, or test andcontrol hybrid ligands/compounds were deposited on the agar in a regularpattern (between 3 and 50 mm spacing) using a standard laboratorypipetting robot (Multiprobe II, Packard, US). FIG. 12 shows an exampleof the analysis performed. Clones that were able to grow on spottingwith GPC 285993 or DMSO alone were discarded. Around 102 clones showedgrowth only on spotting with GPC 285985. These clones were recovered andidentified by DNA sequencing and comprised cDNA clones representinghcdk2 genes as well as other genes.

[0633] To validate the compound specificity of the interaction betweengenes isolated in the above screen, the genes were recloned, and thehalo assay repeated. One unknown gene (denominated GPC-761) was isolatedfour times in the screen described above. One of the isolated plasmidscoding for this gene in vector pACT2 was co-transformed with pBTM118c-DHFR into the yeast strain L40 and a halo assay conducted againstGPC 285985 or GPC 285993 (dissolved in DMSO) or 1 μl DMSO as a control.FIG. 13 demonstrates compound-specific growth of the clone containingGPC-761. Equivalent results were also seen for such validation testsconducted using the hcdk2 genes identified from the above screen.

[0634] Substitution at the Nitrogen in 2-position of the4-oxoindeno[3,2-c]pyrazol group as in GPC 285993 had been proven toabolish all activity towards CDK2 in this substance class (data notshown). The binding of GPC-761 to GPC 285985 but not to then-substituted equivalent GPC 285993 is similar in characteristic to thatof CDK2 binding to these compounds. This demonstrates, that the methodsprovided herein are able to identify a polypeptide binding to auser-specified ligand from a large pool of polypeptides without priorknowledge of the polypeptide.

Example 13 A 3-Hybrid Assay Using Mammalian Cells

[0635] Mammalian cells may possess distinct advantages for performingthe three hybrid assay. They may exhibit better compound intake and mayallow detection of interactions that would not be seen in heterologoushost cells due to their ability to provide machinery/environment forcorrect folding and/or post-translational modifications that may berequired for certain interactions.

[0636] To test the performance of the dimerizing hybrid ligands andmethods of the invention in mammalian cells, the activation of a CATreporter gene using the Mammalian Matchmaker System (Clontech, Cat. No.:K1602-1) was tested. For this purpose, DHFR was cloned into vector pM(Clontech) and GR2 into the vector pVP16 (Clontech) using analogousmethods as described in Example 3; the resulting vectors are termedpM-DHFR and pVP 16-GR2. Standard HeLa cells were transfected withpM3-VP16 and pG5CAT (positive control) or pM-DHFR, pVP16-GR2, andpG5CAT. 24 hours after transfection the medium was exchanged for mediumto which 100 μl/100 ml medium of a 100 μM solution of GPC 285937 in DMSOwas added (FIG. 14A,B) or medium containing the same amount of DMSO(FIG. 14C). 24 hours later the CAT activity was visualized using the CATstaining set (Roche, Cat. No. 1836358). A colored precipitate wasclearly seen in the positive control (FIG. 14A) and in the cellsexpressing the DHFR and GR2 fusions incubated with GPC 285937 (FIG.14B), but no coloured precipitate was seen in the DMSO control (FIG.14C).

[0637] This shows, that the methods of the invention may be transferredto a cell system other than yeast.

Example 14 Methods of Identifying a Ligand for a User-Specified Peptide:a Three-Hybrid Assay System Based on Transcriptional-Based InteractionSystem

[0638] In certain applications, it is advantageous to have methods athand that can identify a small molecule from a pool or library of smallmolecules that is able to bind to a certain first polypeptide P1 ofinterest. To this end, a library of small molecules R1 may be preparedby well established methods of, for example, combinatorial chemistry, orother methods known to the skilled artisan, and subsequently coupled toa second ligand R2 known to bind to a second polypeptide P2 via a(—CH₂—X—CH₂)_(n)-linker to form a library of R1(—CH₂—X—CH₂)_(n)—R2hybrid ligand compounds. Alternatively, a library ofR1(—CH₂—X—CH₂)_(n)—R2 hybrid ligand compounds may be prepared de novo,using steps such as those given in Schemes 1-4 in FIG. 1. However, thisis not meant to limit the scope of the invention to said schemes.Rather, the skilled artisan will, depending on the intended applicationchoose from the large variety of known chemical reactions those bestsuited to generate the library fitting his needs.

[0639] If, for example, without limitation, R2 is chosen to bemethotrexate, the library of hybrid ligand compounds can be used in thefollowing screen: The coding sequence for P1 is amplified from asuitable library or sample known to contain this sequence using primerschosen to be specific for P1, digested, and subcloned into vectorpGAD426c, to give pGAD426c-P1. Cells from yeast strain L40 areco-transformed with pBTM118c-DHFR and pGAD426c-P1. Transformantsreceiving the plasmid are selected on synthetic medium lackingtryptophan and leucine, and individual colonies are regrown in liquidmedium. Microtiter plates are prepared to contain individual or pooledmembers of the library of hybrid ligand compounds at an appropriateconcentration (which may be between 10 mM and 0.1 nM) in SD mediumlacking leu, trp and his. Approximately 1×10⁴, preferably 1×10⁵, morepreferably 1×10⁶, or most preferably 1×10⁷ cells cotransformed withpGAD426c-P1 and pBTM118c-DHFR as prepared above are inoculated into eachwell, and incubated for approximately 1 to 3 days with the solutionscontaining the hybrid ligands.

[0640] Cell growth in the wells is recorded after this growth period.The hybrid ligand compounds known to be present in those wells wheregrowth is detected may subsequently be retested in a validation haloassay as described above in Example 4. In the case of pools of hybridligands, the pools may be fractioned by standard methodologies andindividual hybrid ligands tested in halo assays and subsequentlyidentified by standard methodologies. Where hybrid ligand specificgrowth can be ascertained, the compound linked to methotrexate to formthis hybrid ligand is selected as being able to bind P1.

Example 15 Methods of Identifying a Polypeptide that Binds to aUser-Specified Ligand: a Three-Hybrid Assay System Based on theUbiquitin Split Protein Sensor Technique

[0641] The ubiquitin split protein sensor technique has been used todetect protein interactions in vivo or in vitro. It is generally usefulfor assaying for all kinds of protein-protein interactions, but isparticularly useful in cases where a conventional yeast two-hybrid assayis problematic, i.e. where membrane proteins, transcriptional activatorsor repressors, etc., are involved. Further details of this technique maybe taken, for example, from U.S. Pat. No. 5,585,245, U.S. Pat. No.5,503,977 or Johnsson & Varshavsky (1997) in: The Yeast Two-HybridSystem (Advances in Molecular Biology), Ed. Paul L. Bartel and StanleyFields, Oxford University Press, pp 316-332. Here, we show how theubiquitin split sensor principle may equally be employed in a threehybrid experiment to investigate interactions between proteins and smallmolecules.

[0642] Construction of Vectors for a Three Hybrid Assay System Based onUbiquitin Split Protein Sensor

[0643] Yeast strain JD53 (Dohmen et al., JBC, 1995, 270:18099-109) ischosen for the experiments involving GFP as reporter and detection onWestern Blots, yeast strain L40 is used in experiments where PLV-inducedtranscription of HIS3 is used as readout.

[0644] The plasmid PSDHFR-Cub-PLV, encoding a fusion protein (FIG. 9)comprising Sec62 which facilitates membrane anchoring, DHFR(dihydrofolate reductase), Cub, the C-terminal part of ubiquitin and PLV(chimeric transcription factor: proteinA::lexA::VP16) is constructed asfollows. First, an E. coli folA′ (DHFR) fragment is PCR amplified froman E. coli genomic DNA library (Clontech, Cat# XL4001AB), using primersCR96 and CR97: CR96 5′-GGG GGT CGA CAT GAT CAG TCT GAT TGC GGC GTT AGCG-3′ (SEQ. ID No. 24) CR97 5′-GGG GGC GGC CGC TTA CCG CCG CTC CAG AATCTC AAA G-3′. (SEQ. ID No. 25)

[0645] The sequence of the PCR product is confirmed by sequencing.Second, The PCR product is then digested with SalI and NotI andsubcloned into the Cub-PLV vector (Stagljar et al. (1998) Proc. Natl.Acad. Sci. U.S.A., 95: 5187-92), so that Cub is downstream of theinserted DHFR and upstream of the reporter PLV while all three proteinsare in-frame, yielding plasmid pDHFR-Cub-PLV. Third, the gene encodingthe membrane anchor Sec62 is inserted upstream of DHFR following PCRamplification and sequence confirmation by sequencing of the gene usingprimers with flanking SalI restriction sites. Appropriate PCR primersfor amplification of Sec62 from yeast (S. cerevisiae) genomic DNA areCR98 and CR99: CR98 5′-GAT CGT CGA CAT GGT AGC CGA GCA AAC ACA GGA G-3′(SEQ. ID No. 26) CR99 5′-GAT CGT CGA CGT TTT GTT CGG CTT TTT CAT TGATG-3′. (SEQ. ID No. 27)

[0646] Upon cleavage of the fusion protein after the Cub moiety, PLVwill be released from the fusion and its membrane-anchored location, andtransfers to the nucleus where it activates transcription of genes underthe control of a promoter comprising LexA-binding sites.

[0647] To construct plasmid pDHFR-Cub-GFP, the PLV moiety inpDHFR-Cub-PLV is replaced with a GFP cassette from pCK GFP—S65C usingcompatible restriction sites flanking both cassettes (Reichel, et al.,PNAS, 1996, 93:5888-93). An alternative reporter plasmid,pDHFR-Cub-R-GFP is constructed such that a 20 amino acid leader sequencecontaining lysine is cloned between Cub and GFP such that the firstamino acid of the leader-GFP fragment produced after cleavage of theCub-R peptide bond is an arginine residue.

[0648] Plasmid pNubI-hCDK2 is constructed by digesting the hcdk2 PCRfragment produced in Example 3 with appropriate restriction enzymes andsubcloning the product into plasmid pNubl (Laser et al., PNAS, 2000,97:13732-7).

[0649] To construct a library of plasmids encoding the N-terminal halfof ubiquitin fused to a library of polypeptides, a cDNA library isgenerated from poly A+ RNA isolated from human fetal brain (hFB)(Clontech, CAT# 6525-1) essentially essentially using a commerciallyavailable protocol and reagents (Superscript, Invitrogen, Carlsbad,Calif., USA, CAT. NO. 18248-013) but employing oligo-dT primers forfirst-strand synthesis as follows:

[0650] TT1-A: 5′-TTT TGT ACA TCT AGA TCG CGA GCG GCC GCC CTT TTT TTT TTTTTT TV-3′ (SEQ. ID No. 28)

[0651] with V being A, G, or C at equal molar ratio. The resulting cDNAfragments were subcloned into plasmid pNubl as SalI/NotI restrictionfragments (pADNX-NubIBC; Laser et al., PNAS, 2000, 97:13732-7) to yielda library of plasmids herein termed pNubl-hFB.

[0652] Quantification of the Degree of Cleavage of DHFR-Cub-GFP

[0653] The “bait-Cub-reporter” plasmid pDHFR-Cub-GFP (1 g) isco-transformed with pNubI-hCDK2 into the yeast strain JD53 (Dohmen etal., JBC, 1995, 270:18099-109) by standard techniques (Burke at al.,Methods in yeast genetics: A Cold Spring Harbor Laboratory coursemanual; Cold Spring Harbor Laboratory Press, 2000). Co-transformantscontaining both plasmids are selected on medium lacking leu and trp.Individual colonies are regrown in liquid media and 1×10⁴, preferably1×10⁵, more preferably 1×10⁶, or most preferably 1×10⁷ cells inoculatedinto individual wells of microtitre plates containing SD medium lackingtrp and leu but containing the dimerizing hybrid ligand GPC 285985 at aconcentration of about 50 μM. GPC 285985 was added dissolved in DMSO toa final concentration of approximately 0.1% DMSO, DMSO alone was addedto controls. After 1 to 3 days of incubation at 30° C., cleavage of thereporter moiety GFP from Cub is detected by Western blot analysis usingGFP-specific antibodies (Clontech, Cat. No. 8369-1) and is observed onlyfor cells from the GPC 285985 containing wells. Detection of the cleavedGFP moiety (approx. 29 kDa) is indicative of interaction of the hybridligand and the fusion proteins.

[0654] Repeating the above experiment but using the pDHFR-Cub-R-GFPinstead of pDHFR-Cub-GFP demonstrates loss of GFP activity through N-endrule degradation following its cleavage from Cub brought about byformation of a trimeric complex of the DHFR-Cub-R-GFP and NubI-hCDK2fusion proteins bridged by the hybrid ligand. The fluorescent intensityof GFP in those yeast cells exposed to the hybrid ligand GPC 285985 isreduced compared to those cells exposed only to DMSO. Fluorescentintensity is measured using a standard microtitre plate reader (VictorV, Perkin Elmer) or fluorescence cell-scanning/sorting (FACS) device forexample from Cytomation or Beckton Coulter.

[0655] Quantification of the Degree of Cleavage of Sec62-DHFR-Cub-PL Vby Screening for an Auxotrophic Marker

[0656] The PLV moiety, when synthesized as a Sec62-DHFR-Cub-PLV fusionfrom plasmid PSDHFR-Cub-PLV, is tethered to the ER membrane outside thenucleus and thus, is not available for transcription activation ofreporter genes. Only upon cleavage of the fusion protein after the Cubmoiety, will PLV be released, serving as a transcription factor toactivate reporter genes under the control of the promoter harboring lexAbinding sites inside the nucleus (Stagljar et al. (1998) Proc. Natl.Acad. Sci. U.S.A., 95: 5187-92).

[0657] The “bait-Cub-reporter” plasmid PSDHFR-Cub-PLV (1 μg) isco-transformed with the library of plasmids pNub-hFB (5 μg) into theyeast strain L40 by standard techniques. Transformants are then platedonto 22×22 SD plates prepared with medium lacking leu and trp. After 3days of incubation at 30° C., co-transformants are washed off theplates, mixed and frozen as small aliquots. 2×10⁶ cells are plated on toSD plates lacking trp, leu and his, but containing 50 μM GPC 285985 andincubated for 2-5 days. Only cells containing both plasmids andexhibiting an active HIS3 gene(imidazole-glycerol-phosphate-dehydratase) can survive (first screenpositive). The activation of HIS3 gene is dependent on interactionbetween pNub-hFB, GPC 285985 and pSDHFR-Cub-PLV, which triggersUBP-mediated cleavage of the PLV reporter from the bait fusion protein.The released PLV reporter will then shuttle to the nucleus wheretranscription of the reporter gene (HIS3) is initiated, leading togrowth on SD medium lacking histidine.

[0658] First screen positive clones are picked and tested in ahigh-throughput halo assay analogous to that described in Example 10.Positive clones from this screen are identified by DNA sequencing andinclude clones containing genes expressing CDK2 and other genes.

[0659] A class of false-positives that will occur are proteins containedin the library, that interact with the membrane anchor Sec62. Therefore,a second screen without ligand is performed. Second-screen positives aresolely dependent on the direct interaction between the anchor moietySec62 and the prey from the library. This class of false-positives hasto be subtracted from the positive candidates from the first screen withthe compound.

[0660] This class of false-positives may be eliminated, or at least thenumber of false-positives may be greatly reduced by minimizing themembrane anchor to just the minimal transmembrane domain of Sec62, thatis sufficient to achieve secure membrane anchoring. This minimal domainmay be determined by generating N- and C-terminal deletion constructs ofSec62, using standard laboratory methods.

Example 16 Methods of Identifying a Polypeptide that Binds to aUser-Specified Ligand: a Three-Hybrid Assay System Based on aβ-Lactamase Complementation Technique

[0661] Assays based on the complementation of enzyme fragments fused tointeracting proteins that regenerate enzymatic activity afterdimerization are particularly well suited for monitoring inducibleprotein interactions (reviewed in Rossi, F. M., Blakely, B. T. & Blau,H. M. (2000) Trends Cell. Biol. 10, 119-122). These systemsadvantageously combine low-level expression of test proteins, generationof signal as direct result of the interaction, and enzymaticamplification of this system, resulting in high sensitivity andphysiologically relevant assays. Assays based on enzyme complementationmay be performed in any cell type and in diverse cellular compartmentssuch as the nucleus, secretory vesicles, or plasma membrane. Therefore,these assays perfectly perform where classical 2-hybrid approaches fail(i.e., nuclear localized transcription factors, membrane proteins). Theclass A β-lactamases are particularly attractive candidates for enzymecomplementation assays due to their monomeric nature and relativelysmall size. In addition, β-lactamases have been expressed successfullyin prokaryotic and eukaryotic cells, making this system applicable toboth classes of organisms. A pair of β-lactamase fragments (α197 andω198) was recently identified that complement to produce detectableactivity in bacteria when fused to two helices that form a leucinezipper (see Galarneau et al. (2002) Nature Biotech. 20:619-622 andreferences in Wehrman et al., Proc. Natl. Acad. Sci. U.S.A. (2002),99:3469-3474).

[0662] The identification of the tripeptide, Asn-Gly- Arg (NGR) wasrecently reported, that produced a profound enhancement of β-lactamaseactivity mediated by different protein pairs in bacteria when introducedat the carboxyl terminus of the α197 fragment (Wehrman et al., Proc.Natl. Acad. Sci. U.S.A. (2002), 99:3469-3474).

[0663] It was reasoned that extension of the β-lactamase system intomammalian cells would provide significant advantages over other fragmentcomplementation systems currently used [e.g., β-galactosidase anddihydrofolate reductase (reviewed in Rossi, F. M., Blakely, B. T. &Blau, H. M. (2000) Trends Cell. Biol. 10, 119-122)], because thefragments are small (<19 kDa), there is no endogenous β-lactamaseactivity, and a highly sensitive cell-permeable fluorescent substratehas been developed recently (Zlokarnik et al. (1998) Science-279:84-88).

[0664] Here, we show how the β3-lactamase complementation assayprinciple may equally be employed in a three hybrid experiment toinvestigate interactions between proteins and small molecules. Smallmolecules of the structure R1-Y-R2 are employed to mediate dimerizationbetween a sensor protein P1, here DHFR tightly binding to methotrexate(here, R1), and a test protein P2, here hCDK2 binding to a moiety (R2)of compound GPC285985. The assay may equally be used to monitorinteractions of proteins P2 expressed from a cDNA library with anydimerizing compound R1-Y-R2.

[0665] Construction of Vectors for a Three Hybrid Assay System Based onβ-Lactamase Complementation.

[0666] To create the β-lactamase fusion proteins for expression inmammalian cells, the α197-NGR fragment is amplified by PCR from pUC19(Yanisch-Perron et al., Gene (1985) 33:103-119; commercially availablethrough New England Biolabs, Beverly, Mass., USA) by using the forwardprimer CR100: the forward primer CR100: CR100 5′-GCA

 CCA TGG TGA GTA TTC AAC ATT TCC GTG TCG-3′ (SEQ. ID No. 29) and thereverse primer CR101: CR101 5′-CCG

 TCT ACC ATT TTC GCC AGT TAA TAG TTT GC-3′ (SEQ. ID No. 30)

[0667] The ω198 fragment is amplified by using the primers CR102: theprimers CR102: CR102 5′-CAC

C TAC TTA CTC TAG CTT CCC GGC AAC-3′ (SEQ. ID No. 31) and the reverseprimer CR103: CR103 5′-CAC

 TTA CCA ATG CTT AAT CAG TGA GGC AC-3′ (SEQ. ID No. 32)

[0668] The sequences of the PCR products are confirmed by sequencing.Complementary oligonucleotides containing new restriction sites,including KpnI, HindIII, NotI, and XhoI, are annealed and ligated toplasmids pcDNA3.1/Zeo and pcDNA3.1 linearized with KpnI/XhoI (from bothplasmids a unique HindIII restriction site upstream of KpnI haspreviously been removed by standard procedures). Complementaryoligonucleotides containing restriction site HindIII/NotI and coding inframe for a 15-amino-acid flexible polypeptide linker consisting of(GGGGS)₃ (SEQ. ID No. 33), are hybridized together and ligated intopcDNA3.1/Zeo and pcDNA3.1 linearized with HindIII/NotI. ThePCR-generated products of α197 and ω198 are inserted upstream ordownstream, and in frame with the 15-amino-acid linker, withKpnI/HindIII and NotI/XhoI, respectively. This leads to the creation ofthe construct pcDNA-α97-15α-[N/X]/Zeo and the constructpcDNA-[K/H]-15aa-ω198, respectively. Interacting protein-codingsequences generated by PCR, containing either KpnI/HindIII or NotI/XhoI,are ligated upstream or downstream of the 15-amino-acid linker.

[0669] The plasmid pcDNA_(DHFR)-15aa-ω198, encoding a fusion proteincomprising DHFR, a 15 amino acid linker sequence and the C-terminal partof β-lactamase is constructed as follows. First, an E. coli folA′ (DHFR)fragment is PCR amplified from an E. coli genomic DNA library (Clontech,Cat# XL4001AB), using the primers CR104: the primers CR104: CR104 5′-GGGG

AT GAT CAG TCT GAT TGC GGC GTT AGC G-3′ (SEQ. ID No. 34) and CR105:CR105 5′-GGG G

CC GCC GCT CCA GAA TCT CAA AG-3′. (SEQ. ID No. 35)

[0670] The sequence of the PCR product was confirmed by sequencing.Second, The PCR product is then digested with KpnI and HindIII andsubcloned into pcDNA-[K/H]-15aa-ω198, upstream of the C-terminalβ3-lactamase ω198 fragment, yielding plasmid pcDNA_(DHFR)-15aa-ω198.

[0671] Plasmid pcDNA-α197-15aa-hCDK2 is constructed by digesting a hcdk2PCR fragment produced similarly as the one in Example 3 with appropriaterestriction enzymes and subcloning the product into plasmidpcDNA-α197-15α-[N/X]/Zeo. The cDNA encoding hCDK2 is amplified from ahuman placenta MATCHMAKER cDNA library (Clontech, Cat# HL4025AH,Heidelberg, Germany) by PCR using the primers CR106: the primers CR106:CR106 5′-GG

GA GAA CTT CCA AAA GGT GG-3′ (SEQ. ID No. 36) and CR107: CR107 5′-CG

T CAG AGT CGA AGA TGG GGT AC-3′ (SEQ. ID No. 37)

[0672] The sequence of the PCR product is confirmed by sequencing andthe PCR fragment is cloned into the corresponding NotI and XhoIrestriction sites of plasmid pcDNA-α197-15α-[N/X].

[0673] To construct a library of plasmids encoding the N-terminal halfof β-lactamase (α197), followed by the 15 amino acid linker sequence andfused in frame to a library of polypeptides, a cDNA library is generatedfrom poly A+ RNA isolated from human fetal brain (hFB) (Clontech, CAT#6525-1) essentially using a commercially available protocol and reagents(Superscript, Invitrogen, Carlsbad, Calif., USA, CAT. NO. 18248-013) butemploying oligo-dT primers for first-strand synthesis as follows: CR108:5′-TTT TGT ACA TCT AGA TCG CGA

 CCT TTT TTT TTT TTT TTV-3′ (SEQ. ID No. 38)

[0674] with V being A, G, or C at equal molar ratio. After second strandsynthesis adapters are added to the cDNA by ligation. The adapters areproduced by annealing oligos CR109: annealing oligos CR109: CR109 5′-

 CAC GCG TCC G-3′ (SEQ. ID No. 39) and CR110: CR110 5′-CGG ACG CGTGGC-3′ (SEQ. ID No. 40).

[0675] The resulting cDNA fragments are digested with the restrictionenzyme NotI and subcloned into plasmid pcDNA-α197-15α-[N/X]/Zeo asNotI/XhoI restriction fragments to yield a library of plasmids hereintermed pcDNA-α 197-15aa-hFB.

[0676] Protein-Compound Interaction Measured by the β-LactamaseComplementation Fluorogenic Assay: Human CDK2 Binding to GPC 285985.

[0677] HEK 293 cells are split 24 h before transfection at 1.8×10⁵ cellsonto 15 mm glass coverslips for microscopy (Merck, Germany) in six-welltissue culture plates (Coming Star) in DMEM (Invitrogen, Carlsbad,Calif., USA) enriched with 10% Cosmic calf serum (Hyclone). Cells aretransiently co-transfected with plasmids pcDNADHFR-15aa-o198 andpcDNA-al97-15aa-hCDK2 using Fugene 6 transfection reagent according tothe manufacturer's instructions (Roche Diagnostics). Cells are grown for48 hours selecting for the presence of both plasmids using Zeocin andNeomycin as selective agents. After 48 hours cells are again split at1.8×10⁵ cells. Aliquots of the cells are incubated 48 hours in thepresence of the dimerizing hybrid ligand GPC 285985 at a concentrationof about about 50 μM. GPC 285985 is added dissolved in DMSO to a finalconcentration of approximately 0.1% DMSO, DMSO alone is added tocontrols.

[0678] Cells are washed twice with PBS and once with a physiologicsaline buffer (10 mM HEPES, 6 mM sucrose, 10 mM glucose, 140 mM NaCl, 5mM KCl, 2 mM MgCl2, 2 mM CaCl₂, pH 7.35) before being loaded for onehour at room temperature with 1.5 μM CCF2/AM (Zlokarnik G. et al. (1998)Science 279:84-88). Cells are washed twice with the physiologic salinebuffer. For microscopy, cell fluorescence is observed with FRET filterset XF89-2 (Omega Opticals, Brattleboro, Vt., USA) by excitation of CCF2through a 365 nm filter (50 nm bandpass) with emission observed at 450nm (65 nm bandpass; blue fluorescence) or 535 nm (45 nm bandpass; greenfluorescence). Fluorescence microscopy is conducted on live HEK 293cells with a Leica DMIRB/E (Leica, Germany) inverted microscope and aHCX PL Fluotar 40×/0.75 microscope lens. Images are taken with a colorchilled 3 CCD camera (model C5810; Hamamatsu Photonics, Bridgewater,N.Y.).

[0679] Reconstitution of β-lactamase activity mediated by bridgingbetween DHFR and hCDK2 with GPC 285985 compound, is detected asconversion of the fluorescence emission of the CCFC substrate from abright green fluorescent signal (e.g. DMSO control samples) to a bluefluorescence.

[0680] Compound mediated dimerization can further be detected employingfluorescence spectroscopy or a colorimetric assay format (Zlokarnik G.et al. (1998) Science 279:84-88; and Galameau et al. (2002) NatureBiotech. 20:619-622). Preferably, detection is performed in an automatedfashion, for example in a fluorescence assisted cell sorting (FACS)system.

[0681] Protein-Compound Interaction Measured by the β-LactamaseComplementation Fluorogenic Assay: Searching for Proteins Interactingwith GPC 285985.

[0682] HEK 293 cells are split 24 h before transfection at 1.8×10⁵ cellsonto 15 mm glass coverslips for microscopy (Merck, Germany) in six-welltissue culture plates (Coming Star) in DMEM (Invitrogen, Carlsbad,Calif., USA) enriched with 10% Cosmic calf serum (Hyclone). Cells aretransiently co-transfected with plasmids pcDNA_(DHFR)-15aa-ω198 and thelibrary plasmids pcDNA-oc197-15aa-hFB using Fugene 6 transfectionreagent according to the manufacturer's instructions (RocheDiagnostics). Cells are grown for 48 hours selecting for the presence ofboth plasmids. After 48 hours cells were again split at 1.8×10⁵ cells.Aliquots of the cells were incubated 48 hours in the presence of thedimerizing hybrid ligand GPC 285985 at a concentration of about 50 μM.GPC 285985 was added dissolved in DMSO to a final concentration ofapproximately 0.1% DMSO, DMSO alone was added to controls.

[0683] Fluorescence microscopy and sample preparation was performed asdescribed in the previous section. Reconstitution of β-lactamaseactivity mediated by bridging between DHFR and library clones with GPC285985 compound was detected as conversion of a bright green fluorescentsignal (DMSO control samples) to blue fluorescence. Cells from positivesamples were collected by centrifugation, cell membranes were lysed andplasmid DNA was isolated by standard procedures. Plasmid DNA frompositive clones was analyzed by DNA sequencing.

[0684] Equivalents

[0685] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims. TABLE 2 Inhibitors of kinases Structure Reference

Science 1997, 278(5336): 286-90

Cancer Res. 1999, 59, 2566

Bioorg. Med. Chem Lett. 2002, 12, 221-224

J. Med. Chem. 1998, 41, 3276

Bior. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2000, 10, 223

Bioorg. Med. Chem. Lett. 2001, 11, 9-12

Bioorg. Med. Chem. Lett. 2001, 11, 9-12

Bioog. Med. Chem. Lett. 2000, 10, 2051-2054

WO 01/081311

WO 9917770

Bioorg. Med. Chem. Lett., 2000, 10, 567-569

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

Bioorg. Med. Chem. Lett. 2001, 11, 1401-1405

J. Med. Chem. 2000, 43, 4089-4108

Bioorg. Med. Chem. Lett. 2001, 11, 1922-1914

WO 9920624

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

J. Med. Chem. 2001, 44, 3965-3977

Chem. Biol. 1999, 6, 559 J. Biol, Chem. 1999, 276, 17420, WO 9822103

Nature, 1998, 389, 990

J. Med. Chem. 2001, 44, 4615

J. Med. Chem. 2001, 44, 4615

J. Med. Chem. 2001, 44, 4615

J. Med. Chem. 2001, 44, 4628

J. Med. Chem. 2001, 44, 4628

J. Med. Chem. 2001, 44, 4628

J. Med. Chem. 2001, 44, 4628

Proc. Am. Assoc. Can Res. 1999, 40, 117 & 121

Proc. Am. Assoc. Can. Res. 1999, 90, 69

J. Med. Chem. 1995, 38, 3780

J. Med. Chem. 1995, 38, 3780

J. Med. Chem. 1995, 38, 3780

J. Med. Chem. 1995, 38, 3780

J. Med. Chem. 1997, 40, 3915

J. Med. Chem. 1998, 41, 4365

J. Med. Chem. 1997, 40, 1820

J. Med. Chem. 1998. 41, 4196

Ann. N.Y. Acad. Sci. 1993, 696, 149

J. Med. Chem. 1996, 39, 5215

Proc. Am. Assoc. Can. Res. 1998, 39, 558.

J. Med. Chem. 2001, 44, 3417

J. Biol. Chem. 1997, 272, 29207

WO 01/060816

WO 01/025220

Bioorg. Med. Chem. Lett. 2000, 10, 575-579

Bioorg. Med. Chem. Lett. 2000, 10, 567-569

Bioorg. Med. Chem. Lett. 2000, 10m 945-949

Bioorg. Med. Chem. Lett. 2000, 10, 945-949

Bioorg. Med. Chem. Lett. 2000, 10, 945-949

Bioorg. Med. Chem. Lett. 2000, 10, 945-949

Bioorg. Med. Chem. Lett. 2000, 10, 2047-2050

Bioorg. Med. Chem. Lett. 2000, 10, 2047-2050

Bioorg. Med. Chem. Lett. 2000, 10, 2051-2054

Bioorg. Med. Chem. Lett. 1996, 6, 1759-1764

Bioorg. Med. Chem. Lett. 1996, 6, 1759-1764

J. Biol. Chem. 1991, 266, 15771

J. Med. Chem. 1991, 34, 1896

J. Med. Chem. 1994, 37, 2627

J. Med. Chem. 1999, 41, 2588

Bioorg. Med. Chem. Lett. 2001, 11, 693-696

Bioorg. Med. Chem. Lett. 1998, 8, 3111-3116

Bioorg. Med. Chem. Lett. 2001, 11, 1123-1126

Bioorg. Med. Chem. Lett. 2001, 11, 853-856

Bioorg. Med. Chem. Lett, 2001, 11, 853-856

Bioorg. Med. Chem. Lett. 2001, 11, 853-856

Bioorg. Med. Chem. Lett. 2001, 11, 853-856

Bioorg. Med. Chem. Lett. 2001, 11, 853-856

Bioorg. Med. Chem. Lett. 2001, 11, 849-852

Bioorg. Med. Chem. Lett. 2001, 11, 840-852

Bioorg. Med. Chem. Lett. 2001, 11, 1157-1160

J. Med. Chem. 2001, 44, 2133-2138

WO 01/37835

US 01/051620

WO 01/087846

WO 01/085719

WO 01/085715

J. Am. Chem. Soc. 2001, 123, 11586-11593

WO 01/072711

WO 01/058899

WO 01/010859

WO 00/059901

Bioorg. Med. Chem. Lett. 2000, 10, 2167-2170

WO 00/017203

WO 00/017202

WO 9962890

WO 9917769

J. Am. Chem. Soc. 2001, 123, 11586-11593

Atherosclerosis (Shannon, Ireland) (2002), 160(1), 123-132.

WO 01/066540

WO 01/066539

US 6187799

WO 99/32106

WO 99/32455

WO 99/32436

WO 99/17759

Biochem. Biophys. Res. Commun. 1987, 147, 322

Biochem. Biophys. Res. Commun. 1987, 147, 322

Biochem. Biophys. Res. Commun. 1987, 147, 322

Biochem. Biophys. Res. Commun. 1987, 147, 322

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

Bioorg. Med. Chem. Lett. 2000, 10, 223-226

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We claim:
 1. A hybrid ligand represented by the general formula:R1-Y-R2, wherein: (i) R1 represents a first ligand selected from: asteroid, retinoic acid, beta-lactam antibiotic, cannabinoid, nucleicacid, polypeptide, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel,2,4-diaminopteridine or cyclosporin, or a derivative thereof with minorstructural modifications; (ii) Y represents a polyethylene linker havingthe general formula (CH₂—X—CH₂)_(n), where X represents O, S, SO, orSO₂, and n is an integer from 2 to 25; and, (iii) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide.
 2. The hybrid ligand of claim 1, whereinthe first ligand binds to a polypeptide.
 3. The hybrid ligand of claim2, wherein the binding affinity corresponds to a ligand/polypeptidedissociation constant K_(D) of less than 1 μM.
 4. The hybrid ligand ofclaim 2, wherein the first ligand is capable of forming a covalent bondwith the polypeptide.
 5. The hybrid ligand of claim 1, wherein X is O.6. The hybrid ligand of claim 1, wherein Y is (CH₂—O—CH₂)_(n), where n=2to
 5. 7. The hybrid ligand of claim 1, wherein R1 is dexamethasone. 8.The hybrid ligand of claim 1, wherein R1 is methotrexate, a methotrexatederivative, FK506, an FK506 derivative or a 2,4-diaminopteridinederivative.
 9. The hybrid ligand of claim 1, wherein R1 is methotrexateand Y is (CH₂—O—CH₂)_(n), where n=2 to
 5. 10. The hybrid ligand of claim1, wherein R2 is a ligand selected from: a compound with a knownbiological effect, a compound with an unknown mechanism of action, acompound which binds to more than one polypeptide, a drug candidatecompound, or a compound that binds to an unknown protein.
 11. The hybridligand of claim 1, wherein R2 binds to or inhibits a kinase.
 12. Ahybrid ligand represented by the general formula: R1-Y-R2, wherein: (i)R1 represents a first ligand selected from: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridine orcyclosporin, or a derivative thereof with minor structuralmodifications; (ii) Y represents a linker; and, (iii) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; wherein R2 binds to or inhibits a kinase.13. The hybrid ligand of claim 12, wherein the kinase is a cyclindependent kinase.
 14. The hybrid ligand of claim 12, wherein R2 is aligand selected from Table 2, or a derivative thereof with minorstructural modifications.
 15. The hybrid ligand of claim 12 wherein Yrepresents a polyethylene linker having the general formula(CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n is aninteger from 2 to
 25. 16. A fusion polypeptide, comprising segments P1,Cub-Z, and RM, in an order wherein Cub-Z is closer to the N-terminus ofthe fusion polypeptide than RM, wherein (i) P1 is a ligand bindingpolypeptide that binds to a non-peptide ligand of a hybrid ligand, whichhas the general formula R1-Y-R2, where R1 and R2 are ligands, and Y is alinker, (ii) Cub is a carboxy-terminal subdomain of ubiquitin, (iii) Zis an amino acid residue, (iv) RM is a reporter moiety.
 17. A fusionpolypeptide, comprising segments P1 and Nux, wherein (i) Nux is theamino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, and (ii)P1 is a ligand binding polypeptide that binds to a non-peptide ligand ofa hybrid ligand, which has the general formula R1-Y-R2, where R1 and R2are ligands, R1 is different from R2, and at least one of R1 and R2 isnot a peptide, and Y is a linker.
 18. The fusion polypeptide of claim 16or 17, wherein the non-peptide ligands are: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, FK506, FK506derivative, rapamycin, tetracycline, methotrexate, 2,4-diaminopteridinederivative, novobiocin, maltose, glutathione, biotin, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel,cyclosporin, or a derivative thereof with minor structuralmodifications; or a carbohydrate, polysaccharide, lipid, prostaglandin,acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkylhalide, alkaloid, amine, aromatic hydrocarbon, sulfonate ester,carboxylate acid, aryl halide, ester, phenol, ether, nitrile, carboxylicacid anhydride, amide, quaternary ammonium salt, imine, enamine, amineoxide, cyanohydrin, organocadmium, aldol, organometallic, aromatichydrocarbon, nucleoside, or a nucleotide.
 19. The fusion polypeptide ofclaim 16, wherein Z is a non-methionine amino acid.
 20. The fusionpolypeptide of claim 16, wherein RM is: a polypeptide capable ofemitting light upon excitation, a polypeptide with an enzymaticactivity, a detectable tag or a transcription factor.
 21. The fusionpolypeptide of claim 16, wherein RM is: green fluorescent protein, URA3or PLV.
 22. A nucleic acid encoding the fusion polypeptide of any one ofclaims 16 or
 17. 23. A composition, comprising: (i) a hybrid ligand ofthe general formula R1-Y-R2, where R1 and R2 are ligands, R1 isdifferent from R2 and at least one of R1 and R2 is not a peptide, Y is alinker; and, (ii) at least one of two fusion polypeptides comprising:(a) a first fusion polypeptide comprising segments P2, Cub-Z, and RM, inan order wherein Cub-Z is closer to the N-terminus of the first fusionpolypeptide than RM, wherein P2 is a ligand binding polypeptide that maybind to ligand R1 or R2 of the hybrid ligand, Cub is a carboxy-terminalsubdomain of ubiquitin and RM is a reporter moiety, and Z is an aminoacid residue; (b) a second fusion polypeptide comprising segments Nuxand P1, wherein Nux is the amino-terminal subdomain of a wild-typeubiquitin or a reduced-associating mutant ubiquitin amino-terminalsubdomain, and P1 is a ligand binding polypeptide that may bind toligand R1 or R2 of the hybrid ligand.
 24. A composition, comprising: (i)a hybrid ligand represented by the general formula: R1-Y-R2, wherein:(a) R1 represents a first ligand selected from: a steroid, retinoicacid, beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide,FK506, FK506 derivative, rapamycin, tetracycline, methotrexate,2,4-diaminopteridine derivative, novobiocin, maltose, glutathione,biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone,testosterone, nickel, or cyclosporin, or a derivative thereof with minorstructural modifications; (b) Y represents a polyethylene linker havingthe general formula (CH₂—X—CH₂)_(n), where X represents O, S, SO, orSO₂, and n is an integer from 2 to 25; (c) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; (ii) at least one fusion polypeptideselected from: (a) a first fusion polypeptide comprising: a ligandbinding domain P1 and a domain selected from the group consisting of: aDNA binding domain and a transcriptional activation domain, wherein theligand binding domain binds the first ligand R1; and, (b) a secondfusion polypeptide comprising: a candidate ligand-binding domain P2 forthe user-specified ligand R2 and a domain selected from the groupconsisting of: a DNA binding domain and a transcriptional activationdomain. wherein one of the first and second fusion polypeptides containsa DNA binding domain and the other fusion polypeptide contains atranscription activation domain;
 25. A composition comprising: (i) Ahybrid ligand represented by the general formula: R1-Y-R2, wherein: (a)R1 represents a first ligand selected from: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate,2,4-diaminopteridine derivative, novobiocin, maltose, glutathione,biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone,testosterone, nickel, or cyclosporin or a derivative thereof with minorstructural modifications; (b) Y represents a polyethylene linker havingthe general formula (CH₂—X—CH₂)_(n), where X represents O, S, SO, orSO₂, and n is an integer from 2 to 25; (c) R2 represents auser-specified second ligand different from R1 selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; and (ii) a fusion polypeptide thatincludes: (a) at least one ligand binding domain; and, (b) a functionaldomain heterologous to the ligand binding domain which by itself is notcapable of inducing or allowing the detection of a detectable event, butwhich is capable of inducing or allowing the detection of a detectableevent when brought into proximity of a second functional domain.
 26. Thecomposition of any one of claims 23 to 25, wherein the composition is acomplex.
 27. The composition of any one of claims 23 to 25, wherein thecomposition is provided in an environment chosen from: a cell, acontainer, a kit, a solution or a growth medium.
 28. A method ofidentifying a polypeptide sequence that binds to a user-specified ligandcomprising: (i) providing a hybrid ligand having the general formulaR1-Y-R2, where R1 is a first ligand, R2 is a user-specified ligand, andY is a polyethylene linker having the general formula (CH₂—X—CH₂)_(n),where X represents O, S, SO, or SO₂, and n is, an integer from 2 to 25;(ii) introducing the hybrid ligand into a population of cells, each cellcontaining a hybrid ligand screening system including: (a) a reportergene operably linked to a transcriptional regulatory sequence, saidregulatory sequence including a DNA sequence which binds to a DNAbinding domain; (b) a first chimeric gene encoding a first fusionpolypeptide comprising: a ligand binding domain PI and a domain selectedfrom a DNA binding domain or a transcriptional activation domain,wherein the ligand binding domain binds the first ligand R1; and, (c) asecond chimeric gene encoding a second fusion polypeptide comprising: acandidate ligand-binding domain P2 for the user-specified ligand R2 anda domain selected from a DNA binding domain or a transcriptionalactivation domain; wherein one of the two fusion polypeptides contains aDNA binding domain and the other fusion polypeptide contains atranscription activation domain; (iii) allowing the hybrid ligand tobind the ligand binding domain of the first fusion polypeptide throughthe first ligand R1 and to contact the candidate ligand binding domainof the second fusion polypeptide through the user-specified ligand R2such that, if R2 binds to the candidate ligand binding domain, anincrease in the level of transcription of the reporter gene occurs; (iv)identifying a positive ligand binding cell in which an increase in thelevel of transcription of the reporter gene has occurred; and, (v)identifying the nucleic acid sequence of the second chimeric geneencoding the candidate ligand binding domain that binds to theuser-specified ligand R2, thereby identifying a polypeptide sequencethat binds to a user-specified ligand.
 29. The method of claim 28,wherein the nucleic acid sequence encoding the candidate ligand bindingdomain polypeptide of the second fusion polypeptide is from a libraryselected from: a synthetic oligonucleotide library, a cDNA library, abacterial genomic DNA fragment library, or a eukaryotic genomic DNAfragment library.
 30. The method of claim 28, wherein the first ligandR1 of the hybrid ligand binds to the ligand binding domain P1 with ahigh affinity.
 31. The method of claim 30, wherein the binding affinitycorresponds to a ligand/ligand binding protein dissociation constantK_(D) of less than 1 μM.
 32. The method of claim 28, wherein the firstligand is capable of forming a covalent bond with the ligand bindingdomain P1.
 33. The method of claim 28, wherein X is O.
 34. The method ofclaim 28, wherein Y is (CH₂—O—CH₂)_(n), where n=2 to
 5. 35. The methodof claim 28, wherein R1 is methotrexate, and Y is (CH₂—O—CH₂)_(n), n=2to
 5. 36. The method of claim 28, wherein the reporter gene is selectedfrom: HIS3, LEU2, TRP2, TRP1, ADE2, LYS2, URA3, CYH1, CAN1, lacZ, gfp orCAT.
 37. The method of claim 28, wherein R2 binds to or inhibits akinase.
 38. A method of identifying a polypeptide sequence that binds toa user-specified ligand comprising: (i) providing a hybrid ligand havingthe general formula R1-Y-R2, where R1 is a first ligand, R2 is auser-specified ligand different from R1, at least one of R1 and R2 isnot a peptide, Y is a linker, and wherein R1 binds to or inhibits akinase; (ii) introducing the hybrid ligand into a population of cells,each cell containing a hybrid ligand screening system including: (a) areporter gene operably linked to a transcriptional regulatory sequence,said regulatory sequence including a DNA sequence which binds to a DNAbinding domain; (b) a first chimeric gene encoding a first fusionpolypeptide comprising: a ligand binding domain and a domain selectedfrom the DNA binding domain or a transcriptional activation domain,wherein the ligand binding domain binds the first ligand R1; and, (c) asecond chimeric gene encoding a second fusion polypeptide comprising: acandidate ligand-binding domain for the user-specified ligand R2 and adomain selected from the DNA binding domain or the transcriptionactivation domain; wherein one of the two fusion polypeptides contains aDNA binding domain and the other fusion polypeptide contains atranscription activation domain; (iii) allowing the hybrid ligand tobind the ligand binding domain of the first fusion polypeptide throughthe first ligand R1 and to contact the candidate ligand binding domainof the second fusion polypeptide through the user-specified ligand R2such that, if R2 binds to the candidate ligand binding domain, anincrease in the level of transcription of the reporter gene occurs; (iv)identifying a positive ligand binding cell in which an increase in thelevel of transcription of the reporter gene has occurred; and, (v)identifying the nucleic acid sequence of the second chimeric geneencoding the candidate ligand binding domain that binds to theuser-specified ligand R2, thereby identifying a polypeptide sequencethat binds to a user-specified ligand.
 39. The method of claim 38,wherein the kinase is a cyclin dependent kinase.
 40. The method of claim38, wherein R2 is a compound selected from Table
 2. 41. The method ofclaim 38, wherein Y is (CH₂—X—CH₂)_(n), n=2 to
 25. 42. The method ofclaim 38, wherein R1 represents a first ligand selected from: a steroid,retinoic acid, beta-lactam antibiotic, cannabinoid, nucleic acid,polypeptide, FK506, FK506 derivative, rapamycin, tetracycline,methotrexate, novobiocin, maltose, glutathione, biotin, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone, nickel,2,4-diaminopteridine derivative or cyclosporin, or a derivative thereofwith minor structural modifications.
 43. A method of determining whethera polypeptide P2 and a ligand R2 bind to each other comprising: (i)translationally providing a first ligand-binding polypeptide comprisingsegments P1, Cub-Z, and RM, in an order wherein Cub-Z is closer to theN-terminus of the first ligand-binding polypeptide than RM, and a secondligand-binding polypeptide comprising segments Nux and P2, wherein P1and P2 are polypeptides, Nux is the amino-terminal subdomain of awild-type ubiquitin or a reduced-associating mutant ubiquitinamino-terminal subdomain, Cub is the carboxy-terminal subdomain of awild-type ubiquitin, Z is an amino acid residue and RM is a reportermoiety; (ii) providing a hybrid ligand represented by the generalformula: R1-Y-R2, wherein R1 is a first ligand that binds the firstligand-binding polypeptide at P1, R2 is a second ligand different fromR1, at least one of R1 and R2 is not a peptide, and Y is a linker; (iii)allowing the hybrid ligand to contact the first and secondligand-binding polypeptides; (iv) detecting the degree of cleavage by aubiquitin-specific protease (UBP) of the first ligand-bindingpolypeptide between Cub and Z, wherein an increase of cleavage isindicative of polypeptide P2—ligand R2 binding.
 44. A method ofdetermining whether a polypeptide P1 and a ligand R1 bind to each othercomprising: (i) translationally providing a first ligand-bindingpolypeptide comprising segments P1, Cub-Z, and RM, in an order whereinCub-Z is closer to the N-terminus of the first ligand-bindingpolypeptide than RM, and a second ligand-binding polypeptide comprisingsegments Nux and P2, wherein P1 and P2 are polypeptides, Nux is theamino-terminal subdomain of a wild-type ubiquitin or areduced-associating mutant ubiquitin amino-terminal subdomain, Cub isthe carboxy-terminal subdomain of a wild-type ubiquitin, Z is an aminoacid residue and RM is a reporter moiety; (ii) providing a hybrid ligandrepresented by the general formula: R1-Y-R2, wherein R1 is a firstligand, R2 is a second ligand different from R1 that binds the secondligand-binding polypeptide at P2, at least one of R1 and R2 is not apeptide, and Y is a linker; (iii) allowing the hybrid ligand to contactthe first and second ligand-binding polypeptides; (iv) detecting thedegree of cleavage by a ubiquitin-specific protease (UBP) of the firstligand-binding polypeptide between Cub and Z, wherein an increase ofcleavage is indicative of protein P1—ligand R1 binding.
 45. The methodof claim 43 or 44, wherein said method involves the use of a cellproviding an N-end rule degradation system.
 46. A method of inducing orallowing the detection of a biologically detectable event, comprising:(i) providing at least one cell comprising at least one nucleic acidsequence encoding a fusion polypeptide that includes: (a) at least oneligand binding domain; and, (b) a functional domain which by itself isnot capable of inducing or allowing the detection of the detectableevent; (ii) providing a hybrid ligand of the general formula R1-Y-R2,wherein R1 is different from R2, at least one of R1 and R2 is not apeptide, R1 or R2 represents a ligand that binds to said ligand bindingdomain; Y represents a polyethylene linker having the general formula(CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n is aninteger from 2 to 25; and wherein the binding of said hybrid ligand tosaid ligand binding domain brings the first functional domain intoproximity of a second functional domain, thereby inducing or allowingthe detection of the detectable event; and, (iii) exposing said at leastone cell to an effective amount of said hybrid ligand to bring the firstfunctional domain into proximity of a second functional domain; therebyinducing or allowing the detection of the biologically detectable event.47. A method of identifying a ligand of a user-specified polypeptide,comprising: (i) providing at least one candidate hybrid ligand havingthe general formula R1-Y-R2, where R1 is a first ligand, R2 is acandidate ligand, and Y is a polyethylene linker having the generalformula (CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n isan integer from 2 to 25; (ii) introducing the candidate hybrid ligandinto at least one cell which contains a hybrid ligand screening systemincluding: (a) a reporter gene operably linked to a transcriptionalregulatory sequence, said regulatory sequence including a DNA sequencewhich binds to a DNA binding domain; (b) a first chimeric gene encodinga first fusion polypeptide comprising: a ligand binding domain and adomain selected from: a DNA binding domain or a transcriptionalactivation domain, wherein the ligand binding domain binds the firstligand R1; and, (c) a second chimeric gene encoding a second fusionpolypeptide comprising: a user-specified ligand-binding domain for thecandidate ligand R2 and a domain selected from: a DNA binding domain ora transcription activation domain; wherein one of the two fusionpolypeptides contains a DNA binding domain and the other fusionpolypeptide contains a transcription activation domain; (iii) allowingthe candidate hybrid ligand to bind the ligand binding domain of thefirst fusion polypeptide through the first ligand R1 and to contact theuser-specified ligand binding domain of the second fusion polypeptidethrough the candidate ligand R2 such that, if the user-specified ligandbinding domain binds to the candidate ligand R2, an increase in thelevel of transcription of the reporter gene occurs; (iv) identifying thecandidate hybrid ligand which causes an increase in the level oftranscription of the reporter gene in the cell, thereby identifying thecandidate ligand on the candidate hybrid ligand as a ligand for theuser-specified polypeptide.
 48. A method to investigate the structureactivity relationship of a ligand to a ligand binding domain comprising:(i) providing a hybrid ligand R1-Y-R2, wherein (a) R1 represents a firstligand selected from: a steroid, retinoic acid, beta-lactam antibiotic,cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative,rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione,biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone,testosterone, nickel, 2,4-diaminopteridine derivative or cyclosporin ora derivative thereof with minor structural modifications; (b) Yrepresents a polyethylene linker having the general formula(CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n is aninteger from 2 to 25; and, (c) R2 represents a user-specified secondligand which is different from R1 and is selected from: a peptide,nucleic acid, carbohydrate, polysaccharide, lipid, prostaglandin, acylhalide, alcohol, aldehyde, alkane, alkene, alkyne, alkyl, alkyl halide,alkaloid, amine, aromatic hydrocarbon, sulfonate ester, carboxylateacid, aryl halide, ester, phenol, ether, nitrile, carboxylic acidanhydride, amide, quaternary ammonium salt, imine, enamine, amine oxide,cyanohydrin, organocadmium, aldol, organometallic, aromatic hydrocarbon,nucleoside, or a nucleotide; (ii) providing cells comprising a fusionprotein that includes: (a) at least one ligand binding domain; and, (b)a functional domain heterologous to the ligand binding domain which byitself is not capable of inducing or allowing the detection of adetectable event, but which is capable of inducing or allowing thedetection of a detectable event when brought into proximity of a secondfunctional domain; wherein either a plurality of hybrid ligandscomprising structural variants of said second ligand R2 is provided instep (i), or a plurality of fusion proteins comprising structuralvariants of said ligand binding domain is provided in step (ii); (iii)exposing said cells comprising each fusion protein to an effectiveamount of each hybrid ligand such that the first functional domain maybe brought into proximity of a second functional domain thereby inducingor allowing the detection of a detectable event; (iv) measuring thepresence, amount or activity of any detectable event so induced orallowed in step (iii), thereby investigating the structure activityrelationship between said second ligand and the ligand binding domain.49. The method of claim 48, wherein said first functional domain of (b)is chosen from: a DNA binding domain, a transcription activation domain,a carboxy-terminal subdomain of a wild-type ubiquitin, an amino-terminalsubdomain of a ubiquitin or a reduced-associating mutant ubiquitinamino-terminal subdomain.
 50. The method of any one of claims 28 or 38,further comprising determining the binding affinity of the hybrid ligandto the ligand binding domains P1 and/or P2.
 51. The method of claim 50,wherein the determination of the binding affinity is performed bysurface plasmon resonance.
 52. The method of claim 28 or 38, furthercomprising determining the effects of the hybrid ligand that areindependent of the formation of a trimeric complex comprising the hybridligand, P1 and P2.
 53. The method of any claim 28 or 38, furthercomprising the step of: performing at least one additional separatemethod to confirm that the transcription of the reporter gene isdependent on the presence of the hybrid ligand and the ligand bindingdomains P1 and P2.
 54. The method of claim 53 wherein said additionalseparate method is selected from: a halo growth assay method, amicrotiter plate growth assay, or a fluorescence detection growth assay.55. The method of claim 53 wherein said additional separate method isindividually conducted on greater than about 10, 100, 1000 or 10000different positive ligand binding cell-types identified in step (iv).56. A method to identify a hybrid ligand having the general structureR1-Y-R2 suitable for an in-vivo assay, wherein said assay involves: (i)the use of a hybrid ligand, and (ii) of at least one fusion polypeptidethat includes: (a) at least one ligand binding domain P; and, (b) afunctional domain which by itself is not capable of inducing or allowingthe detection of the detectable event;  and wherein said method involvesthe steps of: (iii) synthesizing a plurality of hybrid ligands R1-Y-R2differing by a plurality of different linkers Y, wherein R1 and R2 aredifferent, and at least one of R1 and R2 is not a peptide; and (iv)testing each hybrid ligand in said plurality of hybrid ligandsindividually for efficacy in inducing or allowing the detection of thedetectable event; and (v) selecting a hybrid ligand with a particularlinker that possesses suitable efficacy in inducing or allowing thedetection of the detectable event.
 57. The method of claim 56 whereinsaid linker has the general structure (CH₂—X—CH₂)_(n), where Xrepresents O, S, SO, or SO₂, and n is an integer from 2 to 25, and theplurality of linkers differ in n.
 58. The method of claim 56 wherein R1represents a first ligand selected from: steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative thereof with minormodifications.
 59. A kit comprising: at least one polynucleotideincluding a DNA fragment linked to a coding sequence for a functionaldomain heterologous to the DNA fragment which by itself is not capableof inducing or allowing the detection of a detectable event, but whichis capable of inducing or allowing the detection of a detectable eventwhen brought into proximity of a second functional domain; and furthercomprising instructions (i) to synthesize a hybrid ligand of generalstructure R1-Y-R2, and (ii) to clone a ligand binding domain into thepolynucleotide, and (iii) to test the binding between the hybrid ligandand the ligand binding domain, wherein R2 is different from R1, one ofR1 and R2 is a non-peptide ligand, and wherein one of R1 and R2 binds toor inhibits a kinase.
 60. A kit comprising at least one polynucleotideincluding a DNA fragment linked to a coding sequence for a functionaldomain heterologous to the DNA fragment which by itself is not capableof inducing or allowing the detection of a detectable event, but whichis capable of inducing or allowing the detection of a detectable eventwhen brought into proximity of a second functional domain; and furthercomprising instructions (i) to synthesize a hybrid ligand of generalstructure R1-Y-R2, and (ii) to clone a ligand binding domain into thepolynucleotide, and (iii) to test the binding between the hybrid ligandand the ligand binding domain, wherein R2 is different from R1, one ofR1 and R2 is a non-peptide ligand, and wherein Y is of the generalstructure (CH₂—X—CH₂)_(n), where X represents O, S, SO, or SO₂, and n isan integer from 2 to
 25. 61. A kit comprising at least onepolynucleotide including a DNA fragment linked to a coding sequence fora functional domain heterologous to the DNA fragment which by itself isnot capable of inducing or allowing the detection of a detectable event,but which is capable of inducing or allowing the detection of adetectable event when brought into proximity of a second functionaldomain; and further comprising instructions (i) to synthesize a hybridligand of general structure R1-Y-R2, and (ii) to clone a ligand bindingdomain into the polynucleotide, and (iii) to test the binding betweenthe hybrid ligand and the ligand binding domain, wherein R2 is differentfrom R1, one of R1 and R2 is a non-peptide ligand, and wherein thefunctional domain is a carboxy-terminal subdomain of ubiquitin or anamino-terminal subdomain of ubiquitin.
 62. A kit comprising: (i) acompound of general structure R1-Y-L, wherein Y is of the generalstructure (CH₂—X—CH₂)_(n), and L is a chemical group that is easilysubstituted by a different chemical group, and (ii) instructions to usethe compound for the synthesis of a hybrid ligand R1-Y-R2 where R1 isdifferent from R2, and at least one of R1 and R2 is not a peptide.
 63. Amethod of doing business comprising: (i) the identification ofpolypeptides binding to a hybrid ligand of general formula R1-Y-R2,wherein Y is of the general structure (CH₂—X—CH₂)_(n), R1 is differentfrom R2, and at least one of R1 and R2 is not a peptide, X═O, S, SO orSO₂, and wherein said polypeptides were previously not known to bind tosuch hybrid ligand, and (ii) providing access to data, nucleic acids orpolypeptides obtained from such identification to another party forconsideration.
 64. The method of claim 63, wherein said identificationof polypeptides is performed using the methods of claims 28, 38, 43 or44.
 65. A method of doing business comprising: (i) the identification ofat least one ligand binding to a user-specified polypeptide by using aplurality of hybrid ligands of general formula R1-Y-R2 differing in atleast one of R1 and R2, wherein R1 and R2 are ligands, R1 is differentfrom R2, at least one of R1 and R2 is not a peptide, Y is of the generalstructure (CH₂—X—CH₂)_(n), X═O, S, SO or SO₂, and wherein said ligandswere previously not known to bind to such polypeptide, and (ii)providing access to data and ligands obtained from such identificationto another party for consideration.
 66. The method of claim 63, whereinsaid identification of ligands is performed using the method of claim47.
 67. A method to identify a polypeptide P2 which binds to a givensmall molecule ligand R2, comprising: (i) immobilizing a polypeptide P1on a matrix, wherein the polypeptide P1 is known to bind a ligand R1,and (ii) contacting said matrix with a hybrid ligand of generalstructure R1-Y-R2, wherein Y is a linker, such that a R2-Y-R1::P1complex is formed on the matrix, and (iii) contacting said complex witha sample comprising at least one candidate polypeptide P2, and (iv)washing said surface such that all constituents of said sample unable tobind to said complex are removed from contact with said complex, and (v)determining whether at least one polypeptide P2 has bound to saidcomplex, and (vi) if a polypeptide P2 was determined to bind to saidcomplex in (v), identifying the polypeptide P2 thereby identifying apolypeptide P2 which binds to a given small molecule ligand R2.
 68. Themethod of claim 67, wherein Y is of the general structure(CH₂—X—CH₂)_(n), and n is an integer from 2 to 25
 69. The method ofclaim 68, wherein X═O, S, SO or SO₂
 70. The method of claim 67, whereinsaid sample is a mixture of several different candidate polypeptides P2.71. The method of claim 67, wherein said sample is a cell extract. 72.The method of claim 67, wherein R1 represents a first ligand chosen fromthe group consisting of: a steroid, retinoic acid, beta-lactamantibiotic, cannabinoid, FK506, FK506 derivative, rapamycin,tetracycline, methotrexate, novobiocin, maltose, glutathione, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone,2,4-diaminopteridine derivative or cyclosporin, or a derivative of oneof the above with minor modifications.
 73. The method of claim 67,wherein P1 is a fusion polypeptide, comprising at least two domainswhich are not found in combination in nature.
 74. The method of claim73, wherein said fusion polypeptide comprises (i) one domain chosen fromthe group consisting of: β-lactamase, a steroid receptor, retinoic acidreceptor, cannabinoid receptor, FKB 12, Tet-R, DHFR, GyrB, maltosebinding protein, glutathione-S-transferase, vitamin D receptor,glucocorticoid receptor, estrogen receptor, progesterone receptor,testosterone receptor, or a fragment of one of the above, which fragmentretains the binding capacity to its respective ligand, and (ii) a seconddomain comprising a tag which allows the immobilization of said fusionprotein on a matrix.
 75. The method of claim 74, wherein said tag ischosen from the group consisting of: a strep tag, FLAG tag, his₆-tag,CBD tag, E tag, GFP tag, GST tag, haemagglutinin tag, Myc tag, T7 tag,Tag 100, V5 tag, Calmodulin binding peptide tag, S tag, Intein/chitinbinding domain tag, Xpress tag, thioredoxin tag or VSV tag.
 76. Themethod of claim 67, wherein P2 is a fusion polypeptide comprising a userspecified fragment and a DNA-binding domain.
 77. The method of claim 76,wherein P2 is bound, through said DNA-binding domain, to a DNA moleculeencoding P2.
 78. The method of claim 77, wherein said DNA binding domainis chosen from the group consisting of: a lac repressor protein, a Repprotein, an NS1 or H-1 protein, a phi-29 terminal protein, a 55Kdprotein, and fragments and derivatives of one of the above, whichfragments and derivatives retain the respective DNA binding activity.79. A composition comprising: (i) a hybrid ligand of general structureR1-Y-R2, wherein Y is a linker, and (ii) a fusion polypeptide,comprising at least two domains which are not found in combination innature, wherein (a) one domain is a user specified polypeptide, and (b)a second domain is a DNA binding domain.
 80. The composition of claim79, wherein Y is of the general structure (CH₂—X—CH₂)_(n), and n is aninteger from 2 to 25
 81. The composition of claim 80, wherein X═O, S, SOor SO₂
 82. The composistion of claim 79, wherein R1 represents a firstligand selected from the group consisting of: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, FK506, FK506 derivative, rapamycin,tetracycline, methotrexate, novobiocin, maltose, glutathione, vitamin D,dexamethasone, estrogen, progesterone, cortisone, testosterone,2,4-diaminopteridine derivative or cyclosporin, or a derivative of oneof the above with minor modifications.
 83. A method of determiningwhether a polypeptide P2 and a ligand R2 bind to each other comprising:(i) translationally providing a first ligand-binding polypeptidecomprising segments P1 and a first fragment of a β-lactamase, and asecond ligand-binding polypeptide comprising segments P2 and a secondfragment of a β-lactamase, wherein said first and second fragments of aβ-lactamase individually possess no β-lactamase activity, and whereinthe enzymatic activity of a β-lactamase is reconstituted when P1 and P2are brought into close spatial proximity; and (ii) providing a hybridligand represented by the general formula: R1-Y-R2, wherein R1 is afirst ligand that binds the first ligand-binding polypeptide at P1, R2is a second ligand different from R1, at least one of R1 and R2 is not apeptide, and Y is a linker; and (iii) allowing the hybrid ligand tocontact the first and second ligand-binding polypeptides; and (iv)detecting the activity of a β-lactamase, wherein an increase inβ-lactamase activity in the presence of said hybrid ligand compared toin its absence is indicative of polypeptide P2—ligand R2 binding,thereby determining whether the polypeptide P2 and a ligand R2 bind toeach other.
 84. The method of claim 83, wherein Y is of the generalstructure (CH₂—X—CH₂)_(n), and n is an integer from 2 to 25
 85. Themethod of claim 84, wherein X═O, S, SO or SO₂
 86. The method of claim83, wherein R1 represents a first ligand selected from the groupconsisting of: steroid, retinoic acid, beta-lactam antibiotic,cannabinoid, nucleic acid, polypeptide, FK506, FK506 derivative,rapamycin, tetracycline, methotrexate, novobiocin, maltose, glutathione,biotin, vitamin D, dexamethasone, estrogen, progesterone, cortisone,testosterone, nickel, 2,4-diaminopteridine derivative or cyclosporin, ora derivative of one of the above with minor modifications.
 87. Acomposition comprising (v) a nucleic acid encoding a polypeptidecomprising a fragment of a β-lactamase and a user specified polypeptide,and (vi) a second nucleic acid encoding a polypeptide comprising asecond fragment of a β-lactamase and a domain chosen from the groupconsiting of: β-lactamase, a steroid receptor, retinoic acid receptor,cannabinoid receptor, FKB 12, Tet-R, DHFR, GyrB, maltose bindingprotein, glutathione-S-transferase, vitamin D receptor, glucocorticoidreceptor, estrogen receptor, progesterone receptor, testosteronereceptor, or a fragment of one of the above, which fragment retains thebinding capacity to its respective ligand, wherein said first and secondfragments of a β-lactamase individually possess no β-lactamase activity,and wherein the enzymatic activity of a β-lactamase is reconstitutedwhen P1 and P2 are brought into close spatial proximity;
 88. Thecomposition of claim 87, further comprising a hybrid ligand of generalstructure R1-Y-R2, wherein Y is a linker.
 89. The composition of claim88, wherein Y is of the general structure (CH₂—X—CH₂)_(n), and n is aninteger from 2 to 25
 90. The composition of claim 89, wherein X═O, S, SOor SO₂
 91. The composition of claim 88, wherein R1 represents a firstligand selected from the group consisting of: steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative of one of the above withminor modifications.
 92. A composition comprising (vii) a polypeptidecomprising a fragment of a β-lactamase and a user specified polypeptide,and (viii) a second polypeptide comprising a second fragment of alactamase and a domain chosen from the group consisting of: β-lactamase,a steroid receptor, retinoic acid receptor, cannabinoid receptor, FKB12, Tet-R, DHFR, GyrB, maltose binding protein,glutathione-S-transferase, vitamin D receptor, glucocorticoid receptor,estrogen receptor, progesterone receptor, testosterone receptor, or afragment of one of the above, which fragment retains the bindingcapacity to its respective ligand, wherein said first and secondfragments of a β-lactamase individually possess no β-lactamase activity,and wherein the enzymatic activity of a β-lactamase is reconstitutedwhen P1 and P2 are brought into close spatial proximity;
 93. Thecomposition of claim 92, further comprising a hybrid ligand of generalstructure R1-Y-R2, wherein Y is a linker.
 94. The composition of claim93, wherein Y is of the general structure (CH₂—X—CH₂)_(n), and n is aninteger from 2 to 25
 95. The composition of claim 94, wherein X═O, S, SOor SO₂
 96. The composition of claim 93, wherein R1 represents a firstligand selected from the group consisting of: a steroid, retinoic acid,beta-lactam antibiotic, cannabinoid, nucleic acid, polypeptide, FK506,FK506 derivative, rapamycin, tetracycline, methotrexate, novobiocin,maltose, glutathione, biotin, vitamin D, dexamethasone, estrogen,progesterone, cortisone, testosterone, nickel, 2,4-diaminopteridinederivative or cyclosporin, or a derivative of one of the above withminor modifications.